Antibodies to tgf-beta

ABSTRACT

The present invention relates to antibody molecules, in particular antibody molecules that bind Transforming Growth Factor beta (TGFβ), and uses thereof. More particularly, the invention relates to antibody molecules that bind and preferably neutralise TGFβ1, TGFβ2 and TGFβ3, so-called “pan-specific” antibody molecules, and uses of such antibody molecules. Preferred embodiments within the present invention are antibody molecules, whether whole antibody (e.g. IgG, such as IgG1 or IgG4) or antibody fragments (e.g. scFv, Fab, dAb).

This application is a divisional application of U.S. application Ser.No. 14/059,241, filed Oct. 21, 2013, which is a divisional applicationof U.S. application Ser. No. 13/615,388, filed Sep. 13, 2012, now U.S.Pat. No. 8,591,901, which is a divisional application of U.S.application Ser. No. 12/724,347, filed Mar. 15, 2010, now U.S. Pat. No.8,383,780, which is a continuation application of U.S. application Ser.No. 11/350,906, filed Feb. 8, 2006, now U.S. Pat. No. 7,723,486, whichclaims the benefit of U.S. Provisional Application 60/651,343 (nowexpired), filed Feb. 8, 2005. The entire disclosure of each of thesereferenced applications is incorporated by reference herein.

A Sequence Listing associated with this application is submittedelectronically via EFS-Web in text format, and is hereby incorporated byreference in its entirety into the specification. The name of the textfile containing the Sequence Listing is001662-0009-107-Sequence-Listing.txt. The text file, created on Apr. 9,2015, is 15689 bytes in size.

TECHNICAL FIELD

The present invention relates to antibody molecules, in particularantibody molecules that bind Transforming Growth Factor beta (TGFβ), anduses thereof. More particularly, the invention relates to antibodymolecules that bind and preferably neutralise TGFβ1, TGFβ2 and TGFβ3,so-called “pan-specific” antibody molecules, and uses of such antibodymolecules.

BACKGROUND

TGFβ was first identified in 1981 (Roberts et al., 1981). In humansthere are three isoforms: TGFβ1, TGFβ2 and TGFβ3 (Swiss Prot accessionnumbers P01137, P08112 and P10600 respectively) which, in theirbiologically active state, are 25 kDa homodimers comprising two 112amino acid monomers joined by an inter-chain disulfide bridge. TGFβ1differs from TGFβ2 by 27, and from TGFβ3 by 22, mainly conservativeamino acid changes. These differences have been mapped on the 3Dstructure of TGFβ determined by X-ray crystallography (Schlunegger etal., 1992; Peer et al., 1996) and the receptor binding regions have beendefined (Griffith et al., 1996; Qian et al., 1996).

Human TGFβs are very similar to mouse TGFβs: human TGFβ1 has only oneamino acid difference from mouse TGFβ1, human TGFβ2 has only three aminoacid differences from mouse TGFβ2 and human TGFβ3 is identical to mouseTGFβ3. As a result, production of antibodies to human TGFβs in mice,including transgenic mice, may be difficult.

TGFβs are multifunctional cytokines that are involved in cellproliferation and differentiation, in embryonic development,extracellular matrix formation, bone development, wound healing,haematopoiesis, and immune and inflammatory responses (Border et al.,1995a). The deregulation of TGFβs leads to pathological processes that,in humans, have been implicated in numerous conditions, for example,birth defects, cancer, chronic inflammatory, autoimmune and fibroticdiseases (Border et al., 1994; Border et al., 1995b).

Studies have been performed in many fibrotic animal models (Border etal., 1995b; Border et al., 1994), using neutralising antibodies asantagonists, for example, glomerulonephritis (Border et al., 1990),neural scarring (Logan et al., 1994), dermal scarring (Shah et al.,1994) and lung fibrosis (Giri et al., 1993). All of the diseasesrepresented by these models represent an unmet need for new therapeuticproducts (Bonewald, 1999; Jackson, 1998). However, the antibodies usedin these and other animal studies have been raised in animals and theirtherapeutic benefit in humans may be limited because of their potentialto induce immunogenic responses and their rapid pharmacokineticclearance (Vaughan et al., 1998). Human antibodies are more desirablefor treatment of TGFβ-

A variety of antibody fragments are known to be able to bind a targetprotein specifically and with good affinity. For example, antibodyfragments comprising only the heavy chain variable (VH) and light chainvariable (VL) domains joined together by a short peptide linker, knownas single chain Fv (scFv), have been used extensively. Human antibodiesneutralising TGFβ1 (CAT-192) or TGFβ2 (CAT-152 or Trabio™) havepreviously been generated (EP 0 945 464, EP 0 853 661, Thompson et al.1999). However, the majority of TGFβ antibodies available in the art arenon-human. Moreover, prior to this invention the only pan-specificmonoclonal antibodies against TGFβ were rodent.

Polyclonal antibodies binding to human TGFβ1 and human TGFβ2 againstboth neutralising and non-neutralising epitopes have been raised inrabbit (Danielpour et al., 1989b; Roberts et al., 1990), chicken (R&DSystems, Minneapolis) and turkey (Danielpour et al., 1989c). Peptidesrepresenting partial TGFβ sequences have been also used as immunogens toraise neutralising polyclonal antisera in rabbits (Border et al., 1990;Flanders et al., 1988). Such non-human, polyclonal antibodies areunsuitable for human therapeutic use.

1D11.16 is a murine pan-specific anti-TGFβ antibody that neutraliseshuman and mouse TGFβ1, TGFβ2 and TGFβ3 in a wide range of in vitroassays (Dasch et al., 1989; Dasch et al., 1996; R&D System product sheetfor MAB1835) and is efficacious in proof-of principle studies in animalmodels of fibrosis (Ling et al., 2003; Miyajima et al., 2000; Schneideret al., 1999; Khanna et al., 1999; Shenkar et al., 1994). However, since1D11.16 is a murine monoclonal antibody (Dasch et al., 1989; Dasch etal., 1996), it is unsuitable for therapeutic use in humans.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 a-c shows the neutralisation (% inhibition) of TGFβ1 (a), TGFβ2(b), or TGFβ3 (c) (10 pM)-induced fibronectin production from NHLF cellsby PET1073G12 germline IgG4 (closed squares) and 1D11.16 (open circles).The closed triangle represents an irrelevant IgG4 tested at the highestconcentration (100 nM). Data are shown as means±SEmean of n experimentsperformed in duplicate. For IC₅₀ values see Table 2.

FIGS. 2 a-c shows the neutralisation (% inhibition) of TGFβ1 (a), TGFβ2(b), or TGFβ3 (c) (10 pM)-induced fibronectin production from NHLF cellsby PET1074B9 germline IgG4 (closed squares) and 1D11.16 (open circles).The closed triangle represents an irrelevant IgG4 tested at the highestconcentration (100 nM). Data are shown as means±SEmean of n experimentsperformed in duplicates.

For IC₅₀ values see Table 2.

FIGS. 3 a-c shows the neutralisation (% inhibition) of TGFβ1 (a), TGFβ2(b), or TGFβ3 (c) (10 pM)-induced fibronectin production from NHLF cellsby PET1287A10 germline IgG4 (closed squares) and 1D11.16 (open circles).The closed triangle represents an irrelevant IgG4 tested at the highestconcentration (100 nM). Data are shown as means±SEmean of n experimentsperformed in duplicate. For IC₅₀ values see Table 2.

SUMMARY OF THE INVENTION

In various aspects of the invention there is provided the subject-matterof the embodiments included below. Further aspects and embodiments ofthe invention are disclosed in the description herein.

The present invention provides specific binding members for TGFβ, inparticular human TGFβ. Specific binding members that are directed toTGFβ1, TGFβ2 and TGFβ3, are particularly provided. Preferred embodimentswithin the present invention are antibody molecules, whether wholeantibody (e.g. IgG, such as IgG1 or IgG4) or antibody fragments (e.g.scFv, Fab, dAb). Antibody antigen-binding regions and antigen bindingsites of antibodies are provided, as are antibody VH and VL domainscontaining such regions. Within VH and VL domains are providedcomplementarity determining regions, CDRs, which may be provided withindifferent framework regions, FR's, to form VH or VL domains as the casemay be. An antigen binding site may consist of an antibody VH domainand/or a VL domain or antigen-binding portions thereof.

In one aspect, the present invention provides a specific binding memberfor human TGFβ, comprising an antigen-binding site of an antibody, anHCDR set, an LCDR set, or both and/or a human antibody VH domain, VLdomain or both.

The set of HCDR1, HCDR2 and HCDR3 may have sequences selected from thefollowing groups:

HCDR1 SEQ ID NO: 3, HCDR2 SEQ ID NO: 4, HCDR3 SEQ ID NO: 5 (referred toherein as the “PET1073G12 set of HCDRs”);HCDR1 SEQ ID NO: 13, HCDR2 SEQ ID NO: 14, HCDR3 SEQ ID NO: 15 (referredto herein as the “PET1074B9 set of HCDRs”);HCDR1 SEQ ID NO: 23, HCDR2 SEQ ID NO: 24, HCDR3 SEQ ID NO: 25 (referredto herein as the “PET1287A10 set of HCDRs”).

The set of LCDR1, LCDR2 and LCDR3 may have sequences selected from thefollowing groups:

LCDR1 SEQ ID NO: 8, LCDR2 SEQ ID NO: 9, LCDR3 SEQ ID NO: 10 (referred toherein as the “PET1073G12 set of LCDRs”);LCDR1 SEQ ID NO: 18, LCDR2 SEQ ID NO: 19, LCDR3 SEQ ID NO: 20 (referredto herein as the “PET1074B9 set of LCDRs”);LCDR1 SEQ ID NO: 28, LCDR2 SEQ ID NO: 29, LCDR3 SEQ ID NO: 30 (referredto herein as the “PET1287A10 set of LCDRs”).

The PET1073G12 set of HCDRs together with the PET1073G12 set of LCDRS isherein referred to as the PET1073G12 set of CDRs.

The PET1074B9 set of HCDRs together with the PET1074B9 set of LCDRS isherein referred to as the PET1074B9 set of CDRs.

The PET1287A10 set of HCDRs together with the PET1287A10 set of LCDRS isherein referred to as the PET1287A10 set of CDRs.

A VH domain comprising a set of HCDRs as disclosed herein is alsoprovided by the present invention, as is separately a VL domaincomprising a set of LCDRs as disclosed herein. Preferably such a VHdomain is paired with such a VL domain, and most preferably the VH andVL domain pairings are the same as in the clones as set out herein.

Further provided by the present invention is a VH domain comprising aset of HCDRs HCDR1, HCDR2 and HCDR3 wherein the set of HCDRs correspondsto that for PET1073G12, PET1074B9 or PET1287A10 with one or two aminoacid substitutions.

Further provided by the present invention is a VL domain comprising aset of LCDRs LCDR1, LCDR2 and LCDR3 wherein the set of CDRs correspondsto that for PET1073G12, PET1074B9 or PET1287A10 with one or two aminoacid substitutions.

A specific binding member comprising an antigen-binding site of anantibody within such a VH and/or VL domain is also provided by thepresent invention.

Following the lead of computational chemistry in applying multivariatedata analysis techniques to the structure/property-activityrelationships (Wold, et al. Multivariate data analysis in chemistry.Chemometrics-Mathematics and Statistics in Chemistry (Ed.: B. Kowalski),D. Reidel Publishing Company, Dordrecht, Holland, 1984 (ISBN90-277-1846-6)) quantitative activity-property relationships ofantibodies can be derived using well-known mathematical techniques suchas statistical regression, pattern recognition and classification(Norman et al. Applied Regression Analysis. Wiley-Interscience; 3rdedition (April 1998) ISBN: 0471170828; Abraham Kandel, Eric Backer.Computer-Assisted Reasoning in Cluster Analysis. Prentice Hall PTR; (May11, 1995), ISBN: 0133418847; Wojtek Krzanowski. Principles ofMultivariate Analysis: A User's Perspective (Oxford Statistical ScienceSeries, No 22 (Paper)). Oxford University Press; (December 2000), ISBN:0198507089; Ian H. Witten, Eibe Frank. Data Mining: Practical MachineLearning Tools and Techniques with Java Implementations. MorganKaufmann; (Oct. 11, 1999), ISBN: 1558605525; David G. T. Denison(Editor), Christopher C. Holmes, Bani K. Mallick, Adrian F. M. Smith.Bayesian Methods for Nonlinear Classification and Regression (WileySeries in Probability and Statistics). John Wiley & Sons; (July 2002),ISBN: 0471490369; Arup K. Ghose, Vellarkad N. Viswanadhan. CombinatorialLibrary Design and Evaluation Principles, Software, Tools, andApplications in Drug Discovery. ISBN: 0-8247-0487-8). The properties ofantibodies can be derived from empirical and theoretical models (forexample, analysis of likely contact residues or calculatedphysicochemical property) of antibody sequence, functional andthree-dimensional structures and these properties can be consideredsingly and in combination.

Analysis of antibodies of known atomic structure has elucidatedrelationships between the sequence and three-dimensional structure ofantibody binding sites (Chothia C. et al. Journal Molecular Biology(1992) 227, 799-817; Al-Lazikani, et al. Journal Molecular Biology(1997) 273(4), 927-948). These relationships imply that, except for thethird region (loop) in VH domains, binding site loops have one of asmall number of main-chain conformations: canonical structures. Thecanonical structure formed in a particular loop has been shown to bedetermined by its size and the presence of certain residues at key sitesin both the loop and in framework regions (Chothia et al. andAl-Lazikani et al., supra).

This study of sequence-structure relationship can be used for predictionof those residues in an antibody of known sequence, but of an unknownthree-dimensional structure, which are important in maintaining thethree-dimensional structure of its CDR loops and hence in maintainingbinding specificity. These predictions can be confirmed by comparison ofthe predictions to the output from lead optimization experiments. In astructural approach, a theoretical model can be created of the antibodymolecule (Chothia, et al. Science, 223, 755-758 (1986)) using any freelyavailable or commercial package such as WAM (Whitelegg, N. R. u. andRees, A. R (2000) Prot. Eng., 12, 815-824). A protein visualisation andanalysis software package such as Insight II (Accelerys, Inc.) or DeepView (Guex, N. and Peitsch, M. C. Electrophoresis (1997) 18, 2714-2723)may then be used to evaluate possible substitutions at each position inthe CDR and FR. This information may then be used to make substitutionslikely to have a minimal or beneficial effect on activity.

The techniques required to make substitutions within amino acidsequences of CDRs, antibody VH or VL domains and specific bindingmembers generally is available in the art. Variant sequences may bemade, with substitutions that may or may not be predicted to have aminimal or beneficial effect on activity, and tested for ability to bindand/or neutralise TGFβ and/or for any other desired property. This isdiscussed further below.

As noted already, the present invention provides specific bindingmembers comprising a defined set of CDRs, in particular the set of CDRsof PET1073G12, PET1074B9 and PET1287A10, and sets of CDRs of PET1073G12,PET1074B9 or PET1287A10 with one or two substitutions within the set ofCDRs.

The relevant set of CDRs is provided within antibody framework regionsor other protein scaffolds, e.g. fibronectin or cytochrome B. Preferablyantibody framework regions are employed.

In a preferred embodiment, the heavy chain utilizes a human V_(H)1family gene. In various embodiments, the heavy chain framework aminoacid sequence contains 1-12, preferably 3-12 and more preferably 3-8amino acid differences compared to the germline amino acid sequence ofthe human V_(H)1 family gene. In some embodiments, the heavy chainframework sequence is the germline sequence. In particularly preferredembodiments, the antibody framework region for the heavy chain may behuman DP-10 (V_(H) 1-69) or human DP-88 (V_(H) 1-e) from the V_(H)1family. Preferably, embodiments utilizing a human DP-10 gene have anon-germline amino acid at residues 27, 78 and 94. In some embodiments,residue 27 is tyrosine, residue 78 is threonine and residue 94 is serineor leucine. In some embodiments, the light chain utilizes a human Vκ3family gene with 1-5, preferably 1-4, more preferably 1-3 amino aciddifferences compared to the germline amino acid sequence. In someembodiments, the light chain framework sequence is the germline humanVκ3 family gene sequence. In particularly preferred embodiments, theframework region for the light chain may be human DPK-22 (A27). In somesuch embodiments, residue 2 is a non-germline amino acid.

In some embodiments residue 2 is a threonine.

In a highly preferred embodiment, a VH domain is provided with the aminoacid sequence of SEQ ID NO: 2, this being termed “PET1073G12 VH domain”,or SEQ ID NO: 12, this being termed “PET1074B9 VH domain”, or SEQ ID NO:22, this being termed “PET1287A10 VH domain”.

In a further highly preferred embodiment, a VL domain is provided withthe amino acid sequence of SEQ ID NO: 7, this being termed “PET1073G12VL domain”, or SEQ ID NO: 17, this being termed “PET1074B9 VL domain”,or SEQ ID NO: 27, this being termed “PET1287A10 VL domain”. A highlypreferred embodiment provided in accordance with the present inventionis composed of the PET1073G12 VH domain, SEQ ID NO: 2, and thePET1073G12 VL domain, SEQ ID NO: 7. Another highly preferred embodimentprovided in accordance with the present invention is composed of thePET1074B9 VH domain, SEQ ID NO: 12, and the PET1074B9 VL domain, SEQ IDNO: 17. Another highly preferred embodiment provided in accordance withthe present invention is composed of the PET1287A10 VH domain, SEQ IDNO: 22, and the PET1287A10 VL domain, SEQ ID NO: 27. These or any otherantibody antigen-binding site provided in accordance with the presentinvention may be provided within any desired antibody molecule format,e.g. scFv, Fab, IgG1, IgG4, dAb etc., as is discussed further elsewhereherein.

In a further highly preferred embodiment, the present invention providesan IgG4 antibody molecule comprising the PET1073G12, PET1074B9 orPET1287A10 VH domain, preferably also comprising the correspondingPET1073G12, PET1074B9 or PET1287A10 VL domain.

Other IgG4 or other antibody molecules comprising the PET1073G12,PET1074B9 or PET1287A10 VH domain, and/or the PET1073G12, PET1074B9 orPET1287A10 VL domain, are provided by the present invention as are otherantibody molecules comprising the PET1073G12, PET1074B9 or PET1287A10set of HCDRs within an antibody VH domain, and/or the PET1073G12,PET1074B9 or PET1287A10 set of LCDRs within an antibody VL domain.

It is convenient to point out here that “and/or” where used herein is tobe taken as specific disclosure of each of the two specified features orcomponents with or without the other. For example “A and/or B” is to betaken as specific disclosure of each of (i) A, (ii) B and (iii) A and B,just as if each is set out individually herein.

As noted, in certain embodiments of the present invention provides aspecific binding member which binds all three isoforms of human TGFβ andwhich comprises the PET1073G12, PET1074B9 or PET1287A10 VH and/or VLdomain or antigen-binding portions of those domains.

In some embodiments, a VH domain is paired with a VL domain to providean antigen binding site. In a preferred embodiment, the PET1073G12 VHdomain (SEQ ID NO: 2) is paired with the PET1073G12 VL domain (SEQ IDNO: 7), so that an antigen binding site is formed comprising both thePET1073G12 VH and VL domains. In a preferred embodiment, the PET1074B9VH domain (SEQ ID NO: 12) is paired with the PET1074B9 VL domain (SEQ IDNO: 17), so that an antigen binding site is formed comprising both thePET1074B9 VH and VL domains. In a preferred embodiment, the PET1287A10VH domain (SEQ ID NO: 22) is paired with the PET1287A10 VL domain (SEQID NO: 27), so that an antibody antigen binding site is formedcomprising both the PET1287A10 VH and VL domains. In other embodiments,the PET1073G12, PET1074B9 or PET1287A10 VH is paired with a VL domainother than the corresponding PET1073G12, PET1074B9 or PET1287A10 VL.Light-chain promiscuity is well established in the art.

Similarly, any set of HCDRs disclosed herein can be provided in a VHdomain that is used as a specific binding member alone or in combinationwith a VL domain. A VH domain may be provided with a set of HCDRs asdisclosed herein, and if such a VH domain is paired with a VL domain,then the VL domain may be provided with a set of LCDRs disclosed herein.A pairing of a set of HCDRs and a set of LCDRs may be as disclosedherein for the PET1073G12, PET1074B9 and PET1287A10 antibodies. Theframework regions of the VH and/or VL domains may be germlineframeworks. Frameworks regions of the heavy chain domain may be selectedfrom the V_(H)-1 family, and a preferred V_(H)-1 framework is DP-10 orDP-88 framework. Framework regions of the light chain may be selectedfrom the Vκ3 family, and a preferred such framework is DPK-22.

One or more CDRs may be taken from a VH or VL domain of which thesequence is disclosed herein and incorporated into a suitable framework.This is discussed further herein. The same applies for other CDRs andsets of CDRs of antibodies as obtained using methods described herein.

An antibody VH domain, an antibody VL domain, a set of HCDRs, a set ofLCDRs, a set of CDRs, one or more HCDRs e.g. an HCDR3, and/or one ormore LCR's e.g. an LCDR3, may be employed in any aspect and embodimentof the present invention as disclosed herein for other molecules, forinstance methods of mutation and selection of antigen binding sites withimproved potency.

Variants of the VH and VL domains and CDRs of the present invention,including those for which amino acid sequences are set out herein, andwhich can be employed in specific binding members for TGFβ can beobtained by means of methods of sequence alteration or mutation andscreening. Such methods are also provided by the present invention.

Variable domain amino acid sequence variants of any of the VH and VLdomains whose sequences are specifically disclosed herein may beemployed in accordance with the present invention, as discussed.Particular variants may include one or more amino acid sequencealterations (addition, deletion, substitution and/or insertion of anamino acid residue), may be less than about 20 alterations, less thanabout 15 alterations, less than about 10 alterations or less than about5 alterations, 4, 3, 2 or 1. Alterations may be made in one or moreframework regions and/or one or more CDRs.

In accordance with further aspects of the present invention there isprovided a human, humanized, chimeric or synthetic specific bindingmember that competes or cross-competes for binding to antigen with anyspecific binding member that both binds the antigen and comprises aspecific antibody antigen-binding region, VH and/or VL domain disclosedherein, set of CDRs or HCDR3 disclosed herein, or a variant of any ofthese. Competition between binding members may be assayed easily invitro, for example using ELISA and/or by tagging a specific reportermolecule to one binding member which can be detected in the presence ofother untagged binding member(s), to enable identification of specificbinding members which bind the same epitope or an overlapping epitope.Cross-competition between binding members may be readily assayed byrunning the reverse assay, e.g., by reversing the tagged and theuntagged binding members to identify pairs that block binding in bothdirections.

Thus, a further aspect of the present invention provides a specificbinding member comprising an antigen-binding site of an antibody whichcompetes or cross-competes with a PET1073G12, PET1074B9 or PET1287A10antibody molecule, in particular PET1073G12, PET1074B9 or PET1287A10scFv and/or IgG4, for binding to TGFβ. In various embodiments, theantibody is a human, humanized, chimeric or synthetic antibody. Infurther aspects, the present invention provides a specific bindingmember comprising an antigen-binding site of a human, humanized,chimeric or synthetic antibody which competes or cross-competes with anantigen-binding site of the present invention for binding to TGFβ,wherein the antigen-binding site of the human, humanized, chimeric orsynthetic antibody is composed of a VH domain and a VL domain, andwherein the VH and VL domains comprise a set of CDRs as disclosedherein.

Given the information disclosed herein, various methods are available inthe art for obtaining human, humanized, chimeric or synthetic antibodiesagainst TGFβ and which may compete or cross-compete with a PET1073G12,PET1074B9 or PET1287A10 antibody molecule, an antibody molecule with aPET1073G12, PET1074B9 or PET1287A10 set of CDRs, an antibody moleculewith a set of PET1073G12, PET1074B9 or PET1287A10 HCDRs, or an antibodymolecule with a set of PET1073G12, PET1074B9 or PET1287A10 LCDRs, forbinding to TGFβ.

In a further aspect, the present invention provides a method ofobtaining one or more specific binding members able to bind TGFβ1, TGFβ2and TGFβ3, the method including bringing into contact a library ofspecific binding members according to the invention and said TGFβs, andselecting one or more specific binding members of the library able tobind all of said TGFβs.

The library may be displayed on the surface of bacteriophage particles,each particle containing nucleic acid encoding the antibody VH variabledomain displayed on its surface, and optionally also a displayed VLdomain if present.

Following selection of specific binding members able to bind the antigenand displayed on bacteriophage particles, nucleic acid may be taken froma bacteriophage particle displaying a said selected specific bindingmember. Such nucleic acid may be used in subsequent production of aspecific binding member or an antibody VH variable domain (andoptionally an antibody VL variable domain) by expression from a nucleicacid with the sequence of nucleic acid taken from a bacteriophageparticle displaying a said selected specific binding member.

An antibody VH domain with the amino acid sequence of an antibody VHdomain of a said selected specific binding member may be provided inisolated form, as may a specific binding member comprising such a VHdomain. Ability to bind all three isoforms of TGFβ may be furthertested, also ability to compete or cross-compete with PET1073G12,PET1074B9 or PET1287A10 (e.g. in scFv format and/or IgG format, e.g.IgG4) for binding to all three human isoforms of TGFβ. Ability toneutralise TGFβ may be tested, as discussed further below.

A specific binding member according to the present invention may bindTGFβ1, TGFβ2 and/or TGFβ3 with the affinity of a PET1073G12, PET1074B9or PET1287A10 antibody molecule, e.g. scFv, or preferably IgG4, or withan affinity that is greater than one of the above molecules. A specificbinding member according to the present invention may neutralise TGFβ1,TGFβ2 and/or TGFβ3 with the potency of a PET1073G12, PET1074B9 orPET1287A10 antibody molecule, e.g. scFv, or preferably PET1073G12,PET1074B9 or PET1287A10 IgG4, or with a potency that is greater than oneof the above molecules.

A specific binding member according to the present invention mayneutralise naturally occurring TGFβ with the potency of a PET1073G12,PET1074B9 or PET1287A10 antibody molecule, e.g. scFv, or preferablyIgG4, or with a potency that is greater than one of the above molecules.Binding affinity and neutralisation potency of different specificbinding members can be compared under appropriate conditions.

A preferred embodiment of the present invention comprises preferablyhuman, humanized, chimeric or synthetic antibodies that neutralisenaturally occurring TGFβ with a potency that is equal to or greater thanthe potency of a TGFβ antigen binding site formed by PET1073G12,PET1074B9 or PET1287A10 VH domain and the corresponding PET1073G12,PET1074B9 or PET1287A10 VL domain.

In addition to antibody sequences, a specific binding member accordingto the present invention may comprise other amino acids, e.g. forming apeptide or polypeptide, such as a folded domain, or to impart to themolecule another functional characteristic in addition to ability tobind antigen. Specific binding members of the invention may carry adetectable label, or may be conjugated to a toxin or a targeting moietyor enzyme (e.g. via a peptidyl bond or linker).

In further aspects, the invention provides an isolated nucleic acidwhich comprises a sequence encoding a specific binding member, VH domainand/or VL domain or CDR according to the present invention, and methodsof preparing a specific binding member, a VH domain and/or a VL domainor CDR of the invention, which methods comprise expressing said nucleicacid under conditions to bring about production of said specific bindingmember, VH domain and/or VL domain, or CDR and recovering it.

Specific binding members according to the invention may be used in amethod of treatment or diagnosis of the human or animal body, such as amethod of treatment (which may include prophylactic treatment) of adisease or disorder in a human patient, which comprises administering tosaid patient an effective amount of a specific binding member of theinvention. Conditions treatable in accordance with the present inventioninclude any in which TGFβ plays a role, especially treatment of fibroticdisease, the modulation of wound healing and the treatment of cancer.

More particularly, specific binding members of the invention are usefulto inhibit the activity of any or all of the three isoforms of humanTGFβ in vitro or in vivo. Such activities include but are not limited toTGFβ mediated signaling, extracellular matrix (ECM) deposition,inhibiting epithelial and endothelial cell proliferation, promotingsmooth muscle proliferation, inducing Type III collagen expression,inducing TGF-β, fibronectin, VEGF and IL-11 expression, binding LatencyAssociated Peptide, tumor induced immunosuppression, promotion ofangiogenesis, activating myofibroblasts, promotion of metastasis andinhibition of NK cell activity.

Specific binding members of the invention also are useful to treatdiseases and conditions that result directly or indirectly from TGFβactivity. Because the specific binding members of the invention arepan-specific, i.e., they bind and inhibit the activity of all threeisoforms of TGFβ, they are particularly advantageous for treatingconditions and diseases that involve two or more TGFβ isoforms (such asinfections and tumors) and severe conditions where inhibiting multipletargets is desirable.

Specific binding members are useful to treat diseases and conditionsincluding, but not limited to, fibrotic diseases (such asglomerulonephritis, neural scarring, dermal scarring, pulmonaryfibrosis, lung fibrosis, radiation induced fibrosis, hepatic fibrosis,myelofibrosis), burns, immune mediated diseases, inflammatory diseases(including rheumatoid arthritis), transplant rejection, cancer,Dupuytren's contracture, and gastric ulcers. They are also useful fortreating, preventing and reducing the risk of occurrence of renalinsufficiencies including but not limited to: diabetic (type I and typeII) nephropathy, radiational nephropathy, obstructive nephropathy,diffuse systemic sclerosis, pulmonary fibrosis, allograft rejection,hereditary renal disease (e.g., polycystic kidney disease, medullarysponge kidney, horseshoe kidney), glomerulonephritis, nephrosclerosis,nephrocalcinosis, systemic lupus erythematosus, Sjogren 's syndrome,Berger's disease, systemic or glomerular hypertension,tubulointerstitial nephropathy, renal tubular acidosis, renaltuberculosis, and renal infarction. In particular, they are useful whencombined with antagonists of the renin-angiotensin-aldosterone systemincluding but not limited to: renin inhibitors, angiotensin-convertingenzyme (ACE) inhibitors, Ang II receptor antagonists (also known as “AngII receptor blockers”), and aldosterone antagonists. Methods for usingthe specific binding members of the present invention in combinationwith such antagonists are set forth in PCT/USO4/13677, the contents ofwhich are incorporated by reference.

Specific binding members of the invention also are useful to treatdiseases and conditions associated with the deposition of ECM, saiddiseases and conditions including, systemic sclerosis, postoperativeadhesions, keloid and hypertrophic scarring, proliferativevitreoretinopathy, glaucoma drainage surgery, corneal injury, cataract,Peyronie's disease, adult respiratory distress syndrome, cirrhosis ofthe liver, post myocardial infarction scarring, post angioplastyrestenosis, scarring after subarachnoid haemorrhage, multiple sclerosis,fibrosis after laminectomy, fibrosis after tendon and other repairs,scarring due to tattoo removal, biliary cirrhosis (including sclerosingcholangitis), pericarditis, pleurisy, tracheostomy, penetrating CNSinjury, eosinophilic myalgic syndrome, vascular restenosis,veno-occlusive disease, pancreatitis and psoriatic arthropathy.

Specific binding members of the invention further are useful inconditions where promotion of re-epithelialization is beneficial. Suchconditions include but are not limited to diseases of the skin, such asvenous ulcers, ischaemic ulcers (pressure sores), diabetic ulcers, graftsites, graft donor sites, abrasions and burns, diseases of the bronchialepithelium, such as asthma, ARDS, diseases of the intestinal epithelium,such as mucositis associated with cytotoxic treatment, oesophagualulcers (reflex disease), stomach ulcers, small intestinal and largeintestinal lesions (inflammatory bowel disease).

Still further uses of specific binding members of the invention are inconditions in which endothelial cell proliferation is desirable, forexample, in stabilizing atherosclerotic plaques, promoting healing ofvascular anastomoses, or in conditions in which inhibition of smoothmuscle cell proliferation is desirable, such as in arterial disease,restenosis and asthma.

Specific binding members of the invention also are useful to enhance theimmune response to macrophage-mediated infections such as those causedby Leishmania spp., Trypanosorna cruzi, Mycobacterium tuberculosis andMycobacterium leprae, as well as the protozoan Toxoplasma gondii, thefungi Histoplasma capsulatum, Candida albicans, Candida parapsilosis,and Cryptococcus neoformans, and Rickettsia, for example, R. prowazekii,R. coronii, and R. tsutsugamushi. They are also useful to reduceimmunosuppression caused, for example, by tumors, AIDS or granulomatousdiseases.

Specific binding members of the invention further are useful in thetreatment of hyperproliferative diseases, such as cancers including butnot limited to breast, prostate, ovarian, stomach, renal, pancreatic,colerectal, skin, lung, cervical and bladder cancers, glioma,mesothelioma, as well as various leukemias and sarcomas, such asKaposi's Sarcoma, and in particular are useful to treat or preventrecurrences or metastases of such tumors. In particular, antagonistspecific binding members of the invention are useful to inhibitcyclosporin-mediated metastases.

It will of course be appreciated that in the context of cancer therapy,“treatment” includes any medical intervention resulting in the slowingof tumour growth or reduction in tumour metastases, as well as partialremission of the cancer in order to prolong life expectancy of apatient.

A further aspect of the present invention provides nucleic acid,generally isolated, encoding an antibody VH variable domain and/or VLvariable domain disclosed herein.

Another aspect of the present invention provides nucleic acid, generallyisolated, encoding a HCDR or LCDR sequence disclosed herein, especiallya HCDR selected from SEQ ID NO:'S: 3, 4, 5, 13, 14, 15, 23, 24 and 25 ora VL CDR selected from SEQ ID NO:'S: 8, 9, 10, 18, 19, 20, 28, 29, 30,most preferably PET1073G12, PET1074B9 or PET1287A10 HCDR3 (SEQ ID NO: 5,15 or 25, respectively). Nucleic acids encoding the PET1073G12,PET1074B9 or PET1287A10 set of CDRs, nucleic acids encoding thePET1073G12, PET1074B9 or PET1287A10 set of HCDRs and nucleic acidsencoding the PET1073G12, PET1074B9 or PET1287A10 set of LCDRs are alsoprovided by the present invention, as are nucleic acids encodingindividual CDRs, HCDRs, LCDRs and sets of the PET1073G12, PET1074B9 orPET1287A10 CDRs, HCDRs, LCDRs.

A further aspect provides a host cell transformed with nucleic acid ofthe invention.

A yet further aspect provides a method of production of an antibody VHvariable domain, the method including causing expression from encodingnucleic acid. Such a method may comprise culturing host cells underconditions for production of said antibody VH variable domain or causingsaid antibody VH domain to be expressed in vivo.

Analogous methods for production of VL variable domains and specificbinding members comprising a VH and/or VL domain are provided as furtheraspects of the present invention.

A method of production may comprise a step of isolation and/orpurification of the product.

A method of production may comprise formulating the product into acomposition including at least one additional component, such as apharmaceutically acceptable excipient.

These and other aspects of the invention are described in further detailbelow.

Particular Embodiments

1. An isolated specific binding member which binds to and neutralizeshuman TGFβ1, TGFβ2 and TGFβ3, comprising an antigen-binding domain of anantibody, wherein said antigen binding domain comprises a set of CDRsHCDR1, HCDR2 and HCDR3, and wherein said antigen binding domain utilizesa human V_(H)1 family gene and wherein said HCDR3 has an amino acidsequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO:15 and SEQ ID NO: 25.2. The specific binding member according to paragraph 1, wherein thehuman V_(H)1 family gene is a human VH1-2 gene.3. The specific binding member according to paragraph 2, where in thehuman VH1-2 gene is a DP-10 or a DP-88 gene.4. The specific binding member according to any one of paragraphs 1 to3, wherein the antigen binding domain further comprises a set of CDRsLCDR1, LCDR2 and LCDR3, and wherein said antigen binding domain utilizesa human Vκ3 family gene and wherein said LCDR3 has an amino acidsequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO:20 and SEQ ID NO: 30.5. The specific binding member according to paragraph 4 wherein theHCDR3 and LCDR3 are selected from the group consisting of:

-   -   (a) SEQ ID NO: 5 and SEQ ID NO: 10, respectively;    -   (b) SEQ ID NO: 15 and SEQ ID NO: 20, respectively; and    -   (c) SEQ ID NO: 25 and SEQ ID NO: 30, respectively.        6. The specific binding member according to paragraph 4, wherein        the human Vκ3 family gene is a human Vκ DPK22 gene.        7. The specific binding member according to paragraph 1, wherein        the HCDR1, HCDR2 and HCDR3 of the VH domain are within a        germline heavy chain framework.        8. The specific binding member according to paragraph 1, wherein        the HCDR1, HCDR2 and HCDR3 of the VH domain are within a        framework that comprises up to 12 mutations from the germline        amino acid sequence.        9. The specific binding member according to paragraph 4, wherein        the LCDR1, LCDR2 and LCDR3 of the Vκ domain are within a        germline heavy chain framework.        10. The specific binding member according to paragraph 4,        wherein the LCDR1, LCDR2 and LCDR3 of the Vκ domain are within a        framework that comprises up to 5 mutations from the germline Vκ        amino acid sequence.        11. An isolated specific binding member that binds to and        neutralizes human TGFβ1, TGFβ2 and TGFβ3, comprising an        antigen-binding domain of an antibody, wherein said antigen        binding domain utilizes a human VH DP-10 gene or a human VH        DP-88 gene and comprises an FR4 amino acid sequence comprising        the amino acid sequence in SEQ ID NO: 31.        12. The specific binding member according to paragraph 11,        wherein said antigen binding domain utilizes a human VH DP-10        gene or a human VH DP-88 gene, and comprises a set of CDRs        HCDR1, HCDR2 and HCDR3, wherein said HCDR3 has an amino acid        sequence selected from the group consisting of SEQ ID NO: 5, SEQ        ID NO: 15 and SEQ ID NO: 25, and further comprises an FR4 amino        acid sequence comprising the amino acid sequence in SEQ ID NO:        31.        13. The specific binding member according to paragraph 11,        wherein the antigen binding domain further utilizes a human Vκ3        family gene and a human Jκ5 gene.        14. The specific binding member according to paragraph 13,        wherein said antigen binding domain utilizing a human Vκ3 family        gene and a human Jκ5 gene comprises a set of CDRs LCDR1, LCDR2        and LCDR3, and wherein said LCDR3 has an amino acid sequence        selected from the group consisting of SEQ ID NO: 10, SEQ ID NO:        20 and SEQ ID NO: 30.        15. An isolated specific binding member which binds to and        neutralises human TGFβ1, TGFβ2 and TGFβ3, comprising an        antigen-binding domain of an antibody, wherein said antigen        binding domain comprises:    -   (a) the HCDR1 of amino acid sequence of SEQ ID NO: 3, HCDR2 of        amino acid sequence of SEQ ID NO: 4, HCDR3 of amino acid        sequence of SEQ ID NO: 5;    -   (b) the HCDR1 of amino acid sequence of SEQ ID NO: 13, HCDR2 of        amino acid sequence of SEQ ID NO: 14, HCDR3 of amino acid        sequence of SEQ ID NO: 15; or    -   (c) the HCDR1 of amino acid sequence of SEQ ID NO: 23, HCDR2 of        amino acid sequence of SEQ ID NO: 24, HCDR3 of amino acid        sequence of SEQ ID NO: 25.        16. The isolated specific binding member according to paragraph        15, wherein the antigen-binding domain further comprises an        antibody VL domain.        17. The isolated specific binding member according to paragraph        15, wherein the antigen-binding domain comprises LCDRs are        selected from the group consisting of:    -   (a) the LCDR1 of amino acid sequence of SEQ ID NO: 8, LCDR2 of        amino acid sequence of SEQ ID NO: 9, LCDR3 of amino acid        sequence of SEQ ID NO: 10;    -   (b) the LCDR1 of amino acid sequence of SEQ ID NO: 18, LCDR2 of        amino acid sequence of SEQ ID NO: 19, LCDR3 of amino acid        sequence of SEQ ID NO: 20; and    -   (c) the LCDR1 of amino acid sequence of SEQ ID NO: 28, LCDR2 of        amino acid sequence of SEQ ID NO: 29, LCDR3 of amino acid        sequence of SEQ ID NO: 30.        18. The isolated specific binding member according to paragraph        15 wherein HCDR1, HCDR2 and HCDR3 of the VH domain are within a        germline heavy chain framework.        19. The isolated specific binding member according to paragraph        18, wherein the germline heavy chain framework is a human V_(H)1        family framework.        20. The isolated specific binding member according to paragraph        15, wherein the HCDR1, HCDR2 and HCDR3 of the VH domain are        within germline human heavy chain framework V_(H)1 DP-10 or        DP-88.        21. The isolated specific binding member according to paragraph        17, wherein the LCDR1, LCDR2 and LCDR3 of the VL domain are        within a germline light chain framework.        22. The isolated specific binding member according to paragraph        21, wherein the germline light chain framework is a human Vκ3        family framework.        23. The isolated specific binding member according to paragraph        21, wherein the antigen binding domain further utilizes a human        Jκ5 gene.        24. The isolated specific binding member according to paragraph        22, wherein the human Vκ3 family gene is a Vκ DPK22 gene.        25. An isolated specific binding member comprising the        PET10730G12 VH domain (SEQ ID NO: 2) with up to 5 mutations, or        an antigen-binding portion thereof.        26. An isolated specific binding member comprising the PET1074B9        VH domain (SEQ ID NO: 12) with up to 5 mutations, or an        antigen-binding portion thereof.        27. An isolated specific binding member comprising the        PET1287A10 VH domain (SEQ ID NO: 22) with up to 5 mutations, or        an antigen-binding portion thereof.        28. The isolated specific binding member according to paragraph        10, further comprising the PET1073G12 VL domain (SEQ ID NO: 7)        with up to 5 mutations, or an antigen-binding portion thereof.        29. The isolated specific binding member according to paragraph        25 further comprising the PET1074B9 VL domain (SEQ ID NO: 17)        with up to 5 mutations, or an antigen-binding portion thereof.        30. The isolated specific binding member according to paragraph        26 further comprising the PET1287A10 VL domain (SEQ ID NO: 27)        with up to 5 mutations, or an antigen-binding portion thereof.        31. An antibody comprising the PET 1073G12 VH domain (SEQ ID        NO: 2) and the PET 1073G12 VL domain (SEQ ID NO: 7).        32. An antibody comprising the PET 1074B9 VH domain (SEQ ID        NO: 12) and the PET 1074B9 VL domain (SEQ ID NO: 17).        33. An antibody comprising the PET 1287A10 VH domain (SEQ ID        NO: 22) and the PET 1287A10 VL domain (SEQ ID NO: 27).        34. The isolated specific binding member according to any one of        paragraphs 1 to 33 that comprises an scFv antibody molecule.        35. The isolated specific binding member according to any one of        paragraphs 1 to 33 that comprises an antibody constant region.        36. The isolated specific binding member according to paragraph        35 wherein the constant region is from an IgG4.        37. A composition comprising a specific binding member according        to any one of paragraphs 1-30 or 34-36.        38. An isolated nucleic acid which comprises a nucleotide        sequence encoding a specific binding member according to any one        of paragraphs 1-30 or 34-36.        39. A host cell transformed with the nucleic acid according to        paragraph 38.        40. A method of producing a specific binding member comprising        culturing a host cell according to paragraph 39 under conditions        for production of said specific binding member and isolating        and/or purifying said specific binding member.        41. The method according to paragraph 40 further comprising        formulating the specific binding member or antibody VH or VL        variable domain into a composition including at least one        additional active component.        42. A method of producing a specific binding member that        specifically binds human TGFβ1, TGFβ2 and TGFβ3, which method        comprises:    -   (a) providing starting nucleic acid encoding a VH domain or a        starting repertoire of nucleic acids each encoding a VH domain,        wherein the VH domain or VH domains comprise germ-line human        framework V_(H)1 DP-10 or DP-88 and either comprise a HCDR1,        HCDR2 and/or HCDR3 to be replaced or lack a HCDR1, HCDR2 and/or        HCDR3 encoding region;    -   (b) combining said starting nucleic acid or starting repertoire        with donor nucleic acid or donor nucleic acids, wherein the        donor nucleic acid encodes a potential HCDR or the donor nucleic        acids encode potential HCDRs, such that said donor nucleic acid        is or donor nucleic acids are inserted into the HCDR1, HCDR2        and/or HCDR3 region in the starting nucleic acid or starting        repertoire, so as to provide a product repertoire of nucleic        acids encoding VH domains;    -   (c) expressing the nucleic acids of said product repertoire to        produce product VH domains;    -   (d) optionally combining said product VH domains with one or        more VL domains;    -   (e) selecting a specific binding member for human TGFβ1, TGFβ2        and TGFβ3, which specific binding member comprises a product VH        domain and optionally a VL domain; and    -   (f) recovering said specific binding member or nucleic acid        encoding it.        43. The method according to paragraph 42, wherein the donor        nucleic acid or donor nucleic acids encode or are produced by        mutation of the amino acid sequence of:    -   (a) HCDR1 of SEQ ID NO: 3, SEQ ID NO: 13 or SEQ ID NO: 23;    -   (b) HCDR2 of SEQ ID NO: 4, SEQ ID NO: 14 or SEQ ID NO: 24;        and/or    -   (c) HCDR3 of SEQ ID NO: 5, SEQ ID NO: 15 or SEQ ID NO: 25.        44. The method according to paragraph 42 or 43, further        comprising fusing the recovered specific binding member to an        antibody constant region.        45. The method according to paragraph 42, wherein the specific        binding member is an scFv antibody molecule.        46. The method according to paragraph 42, wherein the specific        binding member is an Fab antibody molecule.        47. The method according to paragraph 42 wherein the specific        binding member is a whole antibody.        48. A method of treating a disease or disorder selected from the        group consisting of a fibrotic disease, cancer, or an        immune-mediated disease by administering a pharmaceutically        effective amount of a composition according to paragraph 37.        49. Use of a specific binding member according to any one of        paragraphs 1-30 or 34-36 in the manufacture of a medicament for        treatment of a disease or disorder selected from the group        consisting of fibrotic disease, cancer, or an immune-mediated        disease.        50. A method of inhibiting TGFβ1, TGFβ2 or TGFβ3 signalling        comprising the step of contacting TGFβ1, TGFβ2 and TGFβ3 in vivo        with a specific binding member according to any one of        paragraphs 1-30 or 34-36.        51. A method for inhibiting TGFβ1, 2 or 3-mediated fibronectin        production comprising the step of contacting TGFβ1, TGFβ2 and        TGFβ3 with a specific binding member according to any one of        paragraphs 1-30 or 34-36.        52. A method for inhibiting TGFβ1, 2 or 3-mediated VEGF        production comprising the step of contacting TGFβ1, TGFβ2 and        TGFβ3 with a specific binding member according to any one of        paragraphs 1-30 or 34-36.        53. A method for modulating cell proliferation selected from the        group consisting of:    -   (a) reducing TGFβ1, 2 or 3-mediated inhibition of epithelial        cell proliferation;    -   (b) reducing TGFβ1, 2 or 3-mediated inhibition of endothelial        cell proliferation; and    -   (c) inhibiting TGFβ1, 2 or 3-mediated smooth muscle cell        proliferation, comprising the step of contacting cells        expressing TGFβ1, TGFβ2 or TGFβ3 with a specific binding member        according to any one of paragraphs 1-29 or 33-35.        54. A method for inhibiting cyclosporin-induced TGFβ1, 2 or 3        activity comprising the step of contacting TGFβ1, TGFβ2 or TGFβ3        with a specific binding member according to any one of        paragraphs 1-30 or 34-36.        55. A method for increasing NK cell activity comprising the step        of contacting cells expressing TGFβ1, TGFβ2 or TGFβ3 with a        specific binding member according to any one of paragraphs 1-30        or 34-36.        56. A method for inhibiting TGFβ1, 2 or 3-mediated        immunosuppression comprising the step of contacting cells        expressing TGFβ1, TGFβ2 or TGFβ3 with a specific binding member        according to any one of paragraphs 1-30 or 34-36.        57. A method for inhibiting the growth of a TGFβ1, 2 or 3        expressing tumor comprising the step of contacting cells        expressing TGFβ1, TGFβ2 or TGFβ3 with a specific binding member        according to any one of paragraphs 1-30 or 34-36.        58. An isolated specific binding member which specifically binds        to and neutralizes TGFβ1, TGFβ32 and TGFβ3 comprising a germline        heavy chain framework sequence from the human VH1 gene family.        59. The isolated specific binding member according to paragraph        58, wherein the germline heavy chain framework sequence is from        the human DP-10 VH1 gene family.        60. The isolated specific binding member according to paragraph        59, wherein the germline heavy chain framework sequence is from        the human DP-88 VH1 gene family.        61. An isolated specific binding member which specifically binds        to and neutralizes TGFβ1, TGFβ2 and TGFβ3 comprising a germline        light chain sequence from the human Vκ3 gene family.        62. The isolated specific binding member according to paragraph        61, wherein the framework region for the light chain is from        DPK-22.        63. The isolated specific binding member according to paragraph        35 wherein the constant region is from an IgG1.

DETAILED DESCRIPTION Terminology Specific Binding Member

This describes a member of a pair of molecules which have bindingspecificity for one another. The members of a specific binding pair maybe naturally derived or wholly or partially synthetically produced. Onemember of the pair of molecules has an area on its surface, or a cavity,which specifically binds to an area on the surface of, or a cavity in,the other member of the pair of molecules. Thus the members of the pairhave the property of binding specifically to each other. The presentinvention is concerned with specific binding members that bind a targetantigen.

Specific

This may be used to refer to the situation in which a specific bindingmember will not show any significant binding to molecules other than itsspecific binding partner(s) from a given animal. For example, a specificbinding member specific for human TGFβ will not have significant bindingto other non-TGF-β human molecules however it may cross-react with TGF-βfrom other species.

An antigen-binding specific binding member comprises an antigen-bindingsite. For example, a specific binding member may be an antibodymolecule. An antigen binding site may also be provided by means ofarrangement of CDRs on non-antibody protein scaffolds such asfibronectin or cytochrome B, etc. Koide et al., (1998) Journal ofMolecular Biology, 284:1141-1151; Nygren et al. (1997) Current Opinionin Structural Biology, Vol. 7:463-469). Scaffolds for engineering novelbinding sites in proteins have been reviewed in detail by Nygren et al.,supra. Protein scaffolds for antibody mimics are disclosed in WO00/34784 which describes proteins (antibody mimics) that include afibronectin type III domain having at least one randomised loop. Asuitable scaffold into which to graft one or more CDRs, e.g. a set ofHCDRs, may be provided by any domain member of the immunoglobulin genesuperfamily. The scaffold may be a human or non-human protein.

An advantage of a non-antibody protein scaffold is that it may providean antigen-binding site in a conserved framework region that is smallerand/or easier to manufacture than at least some antibody molecules.Small size of a specific binding member may confer useful physiologicalproperties such as an ability to enter cells, penetrate deep intotissues or reach targets within other structures, or to bind withinprotein cavities of the target antigen.

Use of antigen binding sites in non-antibody protein scaffolds isreviewed in Wess, 2004. Typical are proteins having a stable backboneand one or more variable loops, in which the amino acid sequence of theloop or loops is specifically or randomly mutated to create anantigen-binding site having specificity for binding the target antigen.Such proteins include the IgG-binding domains of protein A from S.aureus, transferrin, tetranectin, fibronectin (e.g. 10th fibronectintype III domain) and lipocalins. Other approaches include synthetic“Microbodies” (Selecore GmbH), which are based on cyclotides—smallproteins having intra-molecular disulphide bonds.

In addition to antibody sequences and/or an antigen-binding site, aspecific binding member according to the present invention may compriseother amino acids, e.g. forming a peptide or polypeptide, such as afolded domain, or to impart to the molecule another functionalcharacteristic in addition to ability to bind antigen. Specific bindingmembers of the invention may carry a detectable label, or may beconjugated to a toxin or a targeting moiety or enzyme (e.g. via apeptidyl bond or linker). For example, a specific binding member maycomprise a catalytic site (e.g. in an enzyme domain) as well as anantigen binding site, wherein the antigen binding site binds to theantigen and thus targets the catalytic site to the antigen. Thecatalytic site may inhibit biological function of the antigen, e.g. bycleavage.

Although, as noted, CDRs can be carried by scaffolds such as fibronectinor cytochrome B (Haan & Maggos, 2004 BioCentury, 12(5): A1-A6; Koide etal., supra; Nygren et al., supra), the structure for carrying a CDR or aset of CDRs of the invention will generally be of an antibody heavy orlight chain sequence or substantial portion thereof in which the CDR orset of CDRs is located at a location corresponding to the CDR or set ofCDRs of naturally occurring VH and VL antibody variable domains encodedby rearranged immunoglobulin genes. The structures and locations ofimmunoglobulin variable domains may be determined by reference to Kabat,et al., Sequences of Proteins of Immunological Interest, 4th Edition,U.S. Department of Health and Human Services, 1987, and updates thereof,now available on the Internet (find “Kabat” using any search engine).

Antibody Molecule

This describes an immunoglobulin whether natural or partly or whollysynthetically produced. The term also covers any polypeptide or proteincomprising an antigen binding domain of an antibody. Antibody fragmentswhich comprise an antigen binding domain are molecules such as Fab,scFv, Fv, dAb, Fd and diabodies.

In the genome of a human germline cell, the genetic information forantibody polypeptide chains is contained in multiple gene segmentswithin loci scattered along different chromosomes. Human heavy chains(VH) are encoded on chromosome 14, kappa light chains (Vκ) on chromosome2 and lambda light chains (Vλ) on chromosome 22. During the developmentof B-lymphocytes (antibody producing cells), gene segments in these lociare assembled by recombination leading to the formation of completeantibody heavy or light chain genes (Tonegawa S. Nature, 302, 575-81,1983). Antibody constant regions (VH, Vκ and Vλ) are largely identicalthroughout the human population but considerable diversity exists withinthe variable domains. Such diversity enables the development of manybillions of different antibodies each with specificity for a differenttarget antigen.

Diversity within the variable regions of antibodies is generated inseveral ways. Firstly, at the genetic level there is considerablediversity within antibody variable germline gene sequences.Approximately 50 different VH germlines (Tomlinson I. M. et al., J. Mol.Biol., 227, 776-798, 1992), 35 different Vκ germlines (Tomlinson I. M.et al., EMBO J, 14, 4628-38, 1995) and 30 different Vλ germlines(Williams S. C. & Winter G., Eur. J. Immunol, 23, 1456-61, 1993;Kawasaki K. et al., Genome Res, 7, 250-61, 1997) have been described.Antibodies are generated from different combinations of these germlinegene sequences. Further diversity is then introduced into antibodyvariable domains by processes such as somatic recombination andhypermutation (Tonegawa S. Nature, 302, 575-81, 1983).

Although there is considerable diversity within antibody variablegermline gene sequences, it is possible to group the sequences intofamilies based on sequence homology. The 50 different VH gene sequencescan be grouped into 7 families, the 35 Vκ sequences into 6 families andthe 30 Vλ families into 10 families. The groups vary in size from onemember (VH6 and Vκ4) to up to 21 members (VH3) and the members of eachgroup share a high degree of sequence homology.

Antibodies can be aligned to VH and VL germline sequence databases todetermine their closest germline match and to identify any amino acidchanges introduced by somatic hypermutation. Research has shown that thehuman immune system utilises some germlines (e.g. VH3 DP47) inpreference to others (e.g. VH2) during an immune response (Knappik A. etal., J. Mol. Biol, 296, 57-86, 2000). However, populations of antibodiesisolated by phage display typically utilise a broad range of germlinegenes, even when isolated against a single antigen (Edwards B. et al.,J. Mol Biol, 334, 103-118, 2003).

It is possible to take monoclonal and other antibodies and usetechniques of recombinant DNA technology to produce other antibodies orchimeric molecules which retain the specificity of the originalantibody. Such techniques may involve joining DNA encoding animmunoglobulin variable region to a constant region, or introducing thecomplementarity determining regions (CDRs), of an antibody into theconstant region plus framework regions, of a different immunoglobulin.See, for instance, EP-A-184187, GB 2188638A or EP-A-239400, and a largebody of subsequent literature. A hybridoma or other cell producing anantibody may be subject to genetic mutation or other changes, which mayor may not alter the binding specificity of antibodies produced.

As antibodies can be modified in a number of ways, the term “antibodymolecule” should be construed as covering any specific binding member orsubstance having an antigen-binding site of an antibody with therequired specificity. Thus, this term covers antibody fragments andderivatives, including any polypeptide comprising an antigen bindingdomain, whether natural or wholly or partially synthetic. Chimericmolecules comprising an antigen binding domain of an antibody, orequivalent, fused to another polypeptide are therefore included. Cloningand expression of chimeric antibodies are described in EP-A-0120694 andEP-A-0125023, and a large body of subsequent literature.

Further techniques available in the art of antibody engineering havemade it possible to isolate human and humanised antibodies. For example,human hybridomas can be made as described by Kontermann et al.(Kontermann R and Dubel Stefan; Antibody Engineering, Springer-VerlagNew York, LLC; 2001, ISBN: 3540413545). Phage display, anotherestablished technique for generating specific binding members has beendescribed in detail in many publications such as Kontermann et al.,supra, and WO 92/01047 (discussed further below). Transgenic mice inwhich the mouse antibody genes are inactivated and functionally replacedwith human antibody genes while leaving intact other components of themouse immune system, can be used for isolating human antibodies to humanantigens (Mendez et al., 1997). Human antibodies, either monoclonal orpolyclonal, can also be made in other transgenic animals such as goats,cows, sheep, rabbits, etc.

Synthetic antibody molecules may be created by expression from genesgenerated by means of oligonucleotides synthesized and assembled withinsuitable expression vectors, for example as described by Knappik et al.,supra or Krebs et al., Journal of Immunological Methods 254 2001 67-84.

It has been shown that fragments of a whole antibody can perform thefunction of binding antigens. Examples of binding fragments are (i) theFab fragment consisting of VL, CL, VH and CHI domains; (ii) the Fdfragment consisting of the VH and CHI domains; (iii) the Fv fragmentconsisting of the VL and VH domains of a single antibody; (iv) the dAbfragment (Ward, E. S. et al., Nature 341, 544-546 (1989), McCafferty etal. (1990) Nature, 348, 552-554) which consists of a VH domain; (v)isolated CDR regions; (vi) F(ab′)2 fragments, a bivalent fragmentcomprising two linked Fab fragments (vii) single chain Fv molecules(scFv), wherein a VH domain and a VL domain are linked by a peptidelinker which allows the two domains to associate to form an antigenbinding site (Bird et al., Science, 242, 423-426, 1988; Huston et al.,Proc. Natl. Acad. Sci USA 85, 5879-5883; 1998 viii) bispecific singlechain Fv dimers (PCT/US92/09665) and (ix) “diabodies”, multivalent ormultispecific fragments constructed by gene fusion (WO/13804); F.Holliger et al., Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993). Fv,scFv or diabody molecules may be stabilised by the incorporation ofdisulphide bridges linking the VH and VL domains (Y. Reiter et al.,Nature Biotech, 14, 1239-1245, 1996). Minibodies comprising a scFvjoined to a CH3 domain may also be made (S. Hu et al., Cancer Res., 56,3055-3061, 1996).

A dAb (domain antibody) is a small monomeric antigen-binding fragment ofan antibody, namely the variable region of an antibody heavy or lightchain (Holt et al., 2003). VH dAbs occur naturally in camelids (e.g.camel, llama) and may be produced by immunising a camelid with a targetantigen, isolating antigen-specific B cells and directly cloning dAbgenes from individual B cells. dAbs are also producible in cell culture.Their small size, good solubility and temperature stability makes themparticularly physiologically useful and suitable for selection andaffinity maturation. A specific binding member of the present inventionmay be a dAb comprising a VH or VL domain substantially as set outherein, or a VH or VL domain comprising a set of CDRs substantially asset out herein.

Where bispecific antibodies are to be used, these may be conventionalbispecific antibodies, which can be manufactured in a variety of ways(Holliger, P. and Winter G. Current Opinion Biotechnol. 4, 446-449(1993)), e.g. prepared chemically or from hybrid hybridomas, or may beany of the bispecific antibody fragments mentioned above. Examples ofbispecific antibodies include those of the BiTE™ technology in which thebinding domains of two antibodies with different specificity can be usedand directly linked via short flexible peptides. This combines twoantibodies on a short single polypeptide chain. Diabodies and scFv canbe constructed without an Fc region, using only variable domains,potentially reducing the effects of anti-idiotypic reaction.

Bispecific diabodies, as opposed to bispecific whole antibodies, mayalso be particularly useful because they can be readily constructed andexpressed in E. coli. Diabodies (and many other polypeptides such asantibody fragments) of appropriate binding specificities can be readilyselected using phage display (W094/13804) from libraries. If one arm ofthe diabody is to be kept constant, for instance, with a specificitydirected against TGFβ, then a library can be made where the other arm isvaried and an antibody of appropriate specificity selected. Bispecificwhole antibodies may be made by knobs-into-holes engineering (C. E. B.Ridgeway et al., Protein Eng., 9, 616-621, 1996).

Antigen-Binding Site

This describes the part of a specific binding member, such as anantibody molecule, that contacts and is complementary to part or all ofthe other member in the binding pair, i.e., the antigen. In an antibodymolecule, the antigen-binding site may be referred to as the antibodyantigen-binding site, and comprises the part of the antibody thatspecifically binds to and is complementary to all or part of the targetantigen. Where an antigen is large, an antibody may only bind to aparticular part of the antigen, which part is termed an epitope.

Antigen-Binding Domain

An antigen binding domain is a portion of a specific binding member thatcomprises an antigen-binding site and that binds the target antigen. Insome embodiments, an antigen-binding domain may be provided by one ormore antibody variable domains (e.g. a so-called Fd antibody fragmentconsisting of a VH domain) or antigen-binding portions thereof. In someembodiments, an antigen binding domain comprises an antibody light chainvariable region (VL) and an antibody heavy chain variable region (VH).

Specific binding members may be glycosylated, either naturally or bysystems of various eukaryotic cells (e.g. CHO or NSO (ECACC 85110503)cells), or they may be (for example if produced by expression in aprokaryotic cell) unglycosylated. Glycosylation may also beintentionally altered, for example by inhibiting fucosylation, in orderto increase ADCC activity of the resulting antibody. Accordingly, any ofthe specific binding members of the invention may be expressed so as tominimize or eliminate fucosylation.

In some embodiments, the CDR or VH or VL domain of the invention will beeither identical or highly similar to the specified regions of which thesequence is set out herein. It is contemplated that from 1 to 5,preferably from 1 to 4 or 1 or 2, or 3 or 4, amino acid substitutionsmay be made in the CDR and/or VH or VL domains. VH or VL domains andCDRs and sets of CDRs that are highly similar to those for whichsequences are given herein are encompassed by aspects of the presentinvention, as are those with sequences that are substantially as set outherein.

The structure for carrying a CDR or a set of CDRs of the invention willgenerally be of an antibody heavy or light chain sequence or substantialportion thereof in which the CDR or set of CDRs is located at a locationcorresponding to the CDR or set of CDRs of naturally occurring VH and VLantibody variable domains encoded by rearranged immunoglobulin genes.The structures and locations of immunoglobulin variable domains may bedetermined by reference to Kabat, E. A. et al., Sequences of Proteins ofImmunological Interest, 4th Edition, US Department of Health and HumanServices, 1987, and updates thereof, now available on the Internet (find“Kabat” using any search engine). CDRs are defined according to Kabat etal.

CDRs can also be carried by other scaffolds such as fibronectin orcytochrome B.

Preferably, a CDR amino acid sequence substantially as set out herein iscarried as a CDR in a human variable domain or a substantial portionthereof. The HCDR3 sequences substantially as set out herein representpreferred embodiments of the present invention and it is preferred thateach of these is carried as a HCDR3 in a human heavy chain variabledomain or a substantial portion thereof.

Variable domains employed in the invention may be obtained or derivedfrom any germ-line or rearranged human variable domain, or may be asynthetic variable domain based on consensus or actual sequences ofknown human variable domains. A CDR sequence of the invention (e.g.CDR3) may be introduced into a repertoire of variable domains lacking aCDR (e.g. CDR3), using recombinant DNA technology. Preferred germlineframeworks have been identified already herein.

For example, Marks et al. (Bio/Technology, 1992, 10:779-783) describemethods of producing repertoires of antibody variable domains in whichconsensus primers directed at or adjacent to the 5′ end of the variabledomain area are used in conjunction with consensus primers to the thirdframework region of human VH genes to provide a repertoire of VKvariable domains lacking a CDR2. Marks et al. further describe how thisrepertoire may be combined with a CDR2 of a particular antibody. Usinganalogous techniques, the CDR3-derived sequences of the presentinvention may be shuffled with repertoires of VH or VL domains lacking aCDR3, and the shuffled complete VH or VL domains combined with a cognateVL or VH domain to provide specific binding members of the invention.The repertoire may then be displayed in a suitable host system such asthe phage display system of W092/01047 or any of a subsequent large bodyof literature, including Kay, B. K., Winter, J., and McCafferty, J.(1996) Phage Display of Peptides and Proteins: A Laboratory Manual, SanDiego: Academic Press, so that suitable specific binding members may beselected. A repertoire may consist of from 10⁴ individual membersupwards, for example from 10⁶ to 10⁸ or 10¹⁰ members. Other suitablehost systems include yeast display, bacterial display, T7 display,ribosome display, covalent display and so on.

Analogous shuffling or combinatorial techniques are also disclosed byStemmer (Nature, 1994, 370:389-391), who describes the technique inrelation to a R-lactamase gene but observes that the approach may beused for the generation of antibodies.

A further alternative is to generate novel VH or VL regions carryingCDR-derived sequences of the invention using random mutagenesis of oneor more selected VH and/or VL genes to generate mutations within theentire variable domain. Such a technique is described by Gram et al.(1992, Proc. Natl. Acad. Sci., USA, 89:3576-3580), who used error-pronePCR. In preferred embodiments one or two amino acid substitutions aremade within a set of HCDRs and/or LCDRs.

Another method which may be used is to direct mutagenesis to CDR regionsof VH or VL genes. Such techniques are disclosed by Barbas et al.,(1994, Proc. Natl. Acad. Sci., USA, 91:3809-3813) and Schier et al.(1996, J. Mol. Biol. 263:551-567).

All the above described techniques are known as such in the art and inthemselves do not form part of the present invention. Given thedisclosure provided herein, the skilled person will be able to use suchtechniques to provide specific binding members of the invention usingroutine methodology in the art.

A further aspect of the invention provides a method for obtaining anantigen binding site of an antibody specific for TGFβ antigen, themethod comprising providing by way of addition, deletion, substitutionor insertion of one or more amino acids in the amino acid sequence of aVH domain set out herein a VH domain which is an amino acid sequencevariant of the VH domain, optionally combining the VH domain thusprovided with one or more VL domains, and testing the VH domain or VH/VLcombination or combinations to identify a specific binding member or anantigen binding domain specific for TGFβ and optionally with one or morepreferred properties, preferably ability to neutralise TGFβ activity.Said VL domain may have an amino acid sequence which is substantially asset out herein.

An analogous method may be employed in which one or more sequencevariants of a VL domain disclosed herein are combined with one or moreVH domains.

In a preferred embodiment, PET1073G12, PET1074139 or PET1287A10 VHdomain may be subject to mutation to provide one or more VH domain aminoacid sequence variants which may be combined with one or more VLdomains.

A further aspect of the invention provides a method of preparing aspecific binding member specific for all three isoforms of human TGFβ,which method comprises:

-   -   (a) providing a starting repertoire of nucleic acids encoding a        VH domain which either include a CDR3 to be replaced or lack a        CDR3 encoding region;    -   (b) combining said repertoire with a donor nucleic acid encoding        an amino acid sequence substantially as set out herein for a        HCDR3 such that said donor nucleic acid is inserted into the        CDR3 region in the repertoire, so as to provide a product        repertoire of nucleic acids encoding a VH domain;    -   (c) expressing the nucleic acids of said product repertoire;    -   (d) selecting a specific binding member specific for at least        one isoform of TGFβ; and    -   (e) recovering said specific binding member or nucleic acid        encoding it.

The method may further comprise the steps of carrying out binding assaysand neutralization assays with each of the three isoforms of TGFβ toidentify specific binding members that bind to and neutralize all threeisoforms.

Again, an analogous method may be employed in which a LCDR3 of theinvention is combined with a repertoire of nucleic acids encoding a VLdomain which either include a CDR3 to be replaced or lack a CDR3encoding region.

Similarly, one or more, or all three CDRs may be grafted into arepertoire of VH or VL domains which are then screened for a specificbinding member or specific binding members specific for all isoforms ofhuman TGFβ3.

The VH domain may have a germline sequence, and in preferred embodimentsis DP-10 or DP-88. A VL domain sequence may have a germline sequence,and in preferred embodiments is DPK-22

In a preferred embodiment, one or more of PET1073G12, PET1074B9 orPET1287A10 HCDR1, HCDR2 and HCDR3, or the PET1073G12, PET1074B9 orPET1287A10 set of HCDRs, may be employed, and/or one or more ofPET1073G12, PET1074B9 or PET1287A10 LCDR1, LCDR2 and LCDR3, or thePET1073G12, PET1074B9 or PET1287A10 set of LCDRs.

A substantial portion of an immunoglobulin variable domain will compriseat least the three CDR regions, together with their interveningframework regions. Preferably, the portion will also include at leastabout 50% of either or both of the first and fourth framework regions,the 50% being the C-terminal 50% of the first framework region and theN-terminal 50% of the fourth framework region. Additional residues atthe N-terminal or C-terminal end of the substantial part of the variabledomain may be those not normally associated with naturally occurringvariable domain regions. For example, construction of specific bindingmembers of the present invention made by recombinant DNA techniques mayresult in the introduction of N- or C-terminal residues encoded bylinkers introduced to facilitate cloning or other manipulation steps.Other manipulation steps include the introduction of linkers to joinvariable domains of the invention to further protein sequences includingimmunoglobulin heavy chains, other variable domains (for example in theproduction of diabodies) or protein labels as discussed in more detailelsewhere herein.

Although in a preferred aspect of the invention specific binding memberscomprising a pair of VH and VL domains are preferred, single bindingdomains based on either VH or VL domain sequences form further aspectsof the invention. It is known that single immunoglobulin domains,especially VH domains, are capable of binding target antigens in aspecific manner.

In the case of either of the single specific binding domains, thesedomains may be used to screen for complementary domains capable offorming a two-domain specific binding member able to bind the threeisoforms of human TGFβ.

This may be achieved by phage display screening methods using theso-called hierarchical dual combinatorial approach as disclosed inW092/01047, in which an individual colony containing either an H or Lchain clone is used to infect a complete library of clones encoding theother chain (L or H) and the resulting two-chain specific binding memberis selected in accordance with phage display techniques such as thosedescribed in that reference. This technique is also disclosed in Markset al., ibid.

Specific binding members of the present invention may further compriseantibody constant regions or parts thereof. For example, a VL domain maybe attached at its C-terminal end to antibody light chain constantdomains including human C_(K) or C_(λ) chains, preferably C_(K) chains.Similarly, a specific binding member based on a VH domain may beattached at its C-terminal end to all or part (e.g. a CH1 domain) of animmunoglobulin heavy chain derived from any antibody isotype, e.g. IgG,IgA, IgE and IgM and any of the isotype sub-classes, particularly IgG1and IgG4. IgG4 is preferred. IgG4 is preferred for some applicationsbecause it does not bind complement and does not create effectorfunctions. Where effector function is desired, IgG1 is preferred.Effector function may also be increased by manipulating theglycosylation state of the antibody, such as by decreasing the fucosecontent, by methods which are known in the art. The heavy chain may ormay not have a C-terminal lysine residue. Any synthetic or otherconstant region variant that has these properties and stabilizesvariable regions is also preferred for use in embodiments of the presentinvention.

Also within the invention are heterogeneous preparations of the specificbinding members or antigen-binding fragments thereof disclosed herein.For example, such preparations may be mixtures of antibodies withfull-length heavy chains and heavy chains lacking the C-terminal lysine,with various degrees of glycosylation, with derivatized amino acids,such as cyclization of an N-terminal glutamic acid to form apyroglutamic acid residue and/or with deamidated forms of the heavy andor light chain.

Specific binding members of the invention may be labelled with adetectable or functional label. Detectable labels include radiolabelssuch as ¹³¹I or ⁹⁹TC, which may be attached to antibodies of theinvention using conventional chemistry known in the art of antibodyimaging. Labels also include enzyme labels such as horseradishperoxidase. Labels further include chemical moieties such as biotinwhich may be detected via binding to a specific cognate detectablemoiety, e.g. labelled avidin.

Specific binding members of the present invention are designed to beused in methods of diagnosis or treatment in human or animal subjects,preferably human.

In some embodiments, specific binding members of the invention inhibitTGFβ1, 2 and/or 3 binding to a cell surface TGFβ receptor or receptorcomplex, including but not limited to a complex comprising receptorserine/threonine kinase type I or type II and proteoglycan beta-glycan(TGFβ type III receptor). Accordingly, the invention comprises a methodfor inhibiting TGFβ binding to a cell surface TGFβ receptor or receptorcomplex comprising the step of contacting TGFβ with a specific bindingmember of the invention and detecting inhibition of binding to thereceptor or receptor complex. In various embodiments, inhibition of TGFβbinding to its receptor(s) can be indicated by reduced phosphorylationof TGFβ receptor type I, reduced activation of TGFβ receptor type I,reduced phosphorylation of and/or activation of R-SMAD proteins,particularly SMAD2 and SMAD3, reduced translocation of said SMADproteins to the nucleus, reduced SMAD protein binding to DNA and/ormodulation of the expression of a gene whose expression in said cell orcell type is known to be mediated by TGFβ signaling. Further assays areset forth in the examples.

Accordingly, further aspects of the invention provide methods oftreatment comprising administration of a specific binding member asprovided, pharmaceutical compositions comprising such a specific bindingmember, and use of such a specific binding member in the manufacture ofa medicament for administration, for example in a method of making amedicament or pharmaceutical composition comprising formulating thespecific binding member with a pharmaceutically acceptable excipient.

Specific binding members of the invention may be administered byinjection (for example, subcutaneously, intravenously, intracavity(e.g., after tumor resection), intralesionally, intraperitoneally orintramuscularly), by inhalation, or topically (for example intraocular,intranasal, rectal, into wounds, on skin), or orally. The route ofadministration can be determined by the physicochemical characteristicsof the product, by special considerations for the disease, by dose ordose interval or by the requirement to optimise efficacy or to minimiseside-effects.

It is envisaged that anti-TGFβ treatment will not be restricted toadministration by healthcare professionals. Therefore, subcutaneousinjection, especially using a needle free device may be appropriate.

In accordance with the present invention, compositions provided may beadministered to individuals in need thereof. Administration ispreferably in a “therapeutically effective amount”, this beingsufficient to show benefit to a patient. Such benefit may be at leastamelioration of at least one symptom of a particular disease ordisorder. The actual amount administered, and rate and time-course ofadministration, will depend on the nature and severity of the diseasebeing treated. Prescription of treatment, e.g. decisions on dosage etc,may be determined based on preclinical and clinical studies the designof which is well within the level of skill in the art.

The precise dose will depend upon a number of factors, including whetherthe antibody is for diagnosis or for treatment, the size and location ofthe area to be treated, the precise nature of the antibody (e.g. wholeantibody, fragment or diabody), and the nature of any detectable labelor other molecule attached to the antibody. A typical antibody dose willbe in the range 100 μg to 1 gm for systemic applications, and 1 μg to 1mg for topical applications. Typically, the antibody will be a wholeantibody, preferably the IgG4 isotype. This is a dose for a singletreatment of an adult patient, which may be proportionally adjusted forchildren and infants, and also adjusted for other antibody formats inproportion to molecular weight and activity. Treatments may be repeatedat daily, twice-weekly, weekly, monthly or other intervals, at thediscretion of the physician. In preferred embodiments of the presentinvention, treatment is periodic, and the period between administrationsis about two weeks or more, preferably about three weeks or more, morepreferably about four weeks or more, or about once a month.

Specific binding members of the present invention will usually beadministered in the form of a pharmaceutical composition, which maycomprise at least one component in addition to the specific bindingmember.

Thus pharmaceutical compositions according to the present invention, andfor use in accordance with the present invention, may comprise, inaddition to active ingredient, a pharmaceutically acceptable excipient,carrier, buffer, stabiliser or other materials well known to thoseskilled in the art. Such materials should be non-toxic and should notinterfere with the efficacy of the active ingredient. Such materialscould include, for example, any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible. Someexamples of pharmaceutically acceptable carriers are water, saline,phosphate buffered saline, dextrose, glycerol, ethanol and the like, aswell as combinations thereof. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Additionalexamples of pharmaceutically acceptable substances are wetting agents orminor amounts of auxiliary substances such as wetting or emulsifyingagents, preservatives or buffers, which enhance the shelf life oreffectiveness of the antibody. The precise nature of the carrier orother material will depend on the route of administration, which may beoral, topical, by inhalation or by injection, e.g., intravenous. In apreferred embodiment, the antibody is administered by intravenousinfusion or injection. In another preferred embodiment, the antibody isadministered by intramuscular or subcutaneous injection.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form for example, with an inert diluent or anassimilable edible carrier. A tablet may comprise a solid carrier suchas gelatin or an adjuvant. Liquid pharmaceutical compositions generallycomprise a liquid carrier such as water, petroleum, animal or vegetableoils, mineral oil or synthetic oil. Physiological saline solution,dextrose or other saccharide solution or glycols such as ethyleneglycol, propylene glycol or polyethylene glycol may be included. Thespecific binding member (and other ingredients, if desired) can also beenclosed in a hard or soft shell gelatin capsule, compressed intotablets, or incorporated directly into the subject's diet. For oraltherapeutic administration, the active ingredient can be incorporatedwith excipients and used in the form of ingestible tablets, buccaltablets, troches, capsules, elixirs, suspensions, syrups, wafers, andthe like. To administer a compound of the invention by other thanparenteral administration, it may be necessary to coat the compoundwith, or co-administer the compound with, a material to prevent itsinactivation.

For intravenous injection, or injection at the site of affliction, theactive ingredient will be in the form of a parenterally acceptableaqueous solution which is pyrogen-free and has suitable pK, isotonicityand stability. Those of relevant skill in the art are well able toprepare suitable solutions using, for example, isotonic vehicles such asSodium Chloride Injection, Ringer's Injection, Lactated Ringer'sInjection. Preservatives, stabilisers, buffers, antioxidants and/orother additives may be included, as required.

A composition may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated.

Specific binding members of the present invention may be formulated inliquid, semi-solid or solid forms such as liquid solutions (e.g.,injectable and infusible solutions), dispersions or suspensions,tablets, pills, powders, liposomes and suppositories. The preferred formdepends on the intended mode of administration, therapeutic application,the physicochemical properties of the molecule and the route ofdelivery. Formulations may include excipients, or combinations ofexcipients, for example: sugars, amino acids and surfactants. Liquidformulations may include a wide range of antibody concentrations and pH.Solid formulations may be produced by lyophilization, spray drying, ordrying by supercritical fluid technology, for example.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the specific binding memberin the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying that yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof. The proper fluidity of a solution canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prolonged absorption of injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

In certain embodiments, the antibody compositions active compound may beprepared with a carrier that will protect the antibody against rapidrelease, such as a controlled release formulation, including implants,transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems (J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978).

The present invention provides a method comprising causing or allowingbinding of a specific binding member as provided herein to TGFβ. Asnoted, such binding may take place in vivo, e.g. followingadministration of a specific binding member, or nucleic acid encoding aspecific binding member, to a patient or it may take place in vitro, forexample in ELISA, Western blotting, immunocytochemistry,immuno-precipitation, affinity chromatography, or cell based assays, orin ex vivo based therapeutic methods (e.g., methods in which cells orbodily fluids are contacted ex vivo with a specific binding memberaccording to the invention and then administered to a patient.

The amount of binding of specific binding member to TGFβ may bedetermined. Quantitation may be related to the amount of the antigen ina test sample, which may be of diagnostic interest.

A kit comprising a specific binding member or antibody moleculeaccording to any aspect or embodiment of the present invention is alsoprovided as an aspect of the present invention. In a kit of theinvention, the specific binding member or antibody molecule may belabelled to allow its reactivity in a sample to be determined, e.g. asdescribed further below. Components of a kit are generally sterile andin sealed vials or other containers. Kits may be employed in diagnosticanalysis or other methods for which antibody molecules are useful. A kitmay contain instructions for use of the components in a method, e.g. amethod in accordance with the present invention. Ancillary materials toassist in or to enable performing such a method may be included within akit of the invention.

The reactivities of antibodies in a sample may be determined by anyappropriate means. Radioimmunoassay (RIA) is one possibility.Radioactive labelled antigen is mixed with unlabelled antigen (the testsample) and allowed to bind to the antibody. Bound antigen is physicallyseparated from unbound antigen and the amount of radioactive antigenbound to the antibody determined. The more antigen there is in the testsample the less radioactive antigen will bind to the antibody. Acompetitive binding assay may also be used with non-radioactive antigen,using antigen or an analogue linked to a reporter molecule. The reportermolecule may be a fluorochrome, phosphor or laser dye with spectrallyisolated absorption or emission characteristics. Suitable fluorochromesinclude fluorescein, rhodamine, phycoerythrin and Texas Red. Suitablechromogenic dyes include diaminobenzidine.

Other reporters include macromolecular colloidal particles orparticulate material such as latex beads that are coloured, magnetic orparamagnetic, and biologically or chemically active agents that candirectly or indirectly cause detectable signals to be visually observed,electronically detected or otherwise recorded. These molecules may beenzymes which catalyse reactions that develop or change colours or causechanges in electrical properties, for example. They may be molecularlyexcitable, such that electronic transitions between energy states resultin characteristic spectral absorptions or emissions. They may includechemical entities used in conjunction with biosensors. Biotin/avidin orbiotin/streptavidin and alkaline phosphatase detection systems may beemployed. The signals generated by individual antibody-reporterconjugates may be used to derive quantifiable absolute or relative dataof the relevant antibody binding in samples (normal and test).

The present invention also provides the use of a specific binding memberas above for measuring antigen levels in a competition assay, that is tosay a method of measuring the level of antigen in a sample by employinga specific binding member as provided by the present invention in acompetition assay. This may be where the physical separation of boundfrom unbound antigen is not required. Linking a reporter molecule to thespecific binding member so that a physical or optical change occurs onbinding is one possibility. The reporter molecule may directly orindirectly generate detectable, and preferably measurable, signals. Thelinkage of reporter molecules may be directly or indirectly, covalently,e.g. via a peptide bond or non-covalently. Linkage via a peptide bondmay be as a result of recombinant expression of a gene fusion encodingantibody and reporter molecule.

The present invention also provides for measuring levels of antigendirectly, by employing a specific binding member according to theinvention for example in a biosensor system.

The mode of determining binding is not a feature of the presentinvention and those skilled in the art are able to choose a suitablemode according to their preference and general knowledge.

As noted, in various aspects and embodiments, the present inventionextends to a human, humanized, chimeric or synthetic specific bindingmember which competes for binding to TGFβ (TGFβ1, 2 and/or 3) with anyspecific binding member defined herein, e.g. PET1037GR, PET1074B9 orPET1287A10 IgG4. Competition or cross-competition between bindingmembers may be assayed easily in vitro, for example by tagging aspecific reporter molecule to one binding member which can be detectedin the presence of other untagged binding member(s), to enableidentification of specific binding members which bind the same epitopeor an overlapping epitope.

Competition may be determined for example using ELISA in which TGFβ isimmobilised to a plate and a first tagged binding member (the referencebinding member) along with one or more other untagged binding members isadded to the plate. Presence of an untagged binding member that competeswith the tagged binding member is observed by a decrease in the signalemitted by the tagged binding member.

In testing for competition a peptide fragment of the antigen may beemployed, especially a peptide including an epitope of interest. Apeptide having the epitope sequence plus one or more amino acids ateither end may be used. Such a peptide may be said to “consistessentially” of the specified sequence. Specific binding membersaccording to the present invention may be such that their binding forantigen is inhibited by a peptide with or including the sequence given.In testing for this, a peptide with either sequence plus one or moreamino acids may be used.

Specific binding members which bind a specific peptide may be isolatedfor example from a phage display library by panning with the peptide(s).

The present invention further provides an isolated nucleic acid encodinga specific binding member of the present invention. Nucleic acid mayinclude DNA and/or RNA. In a preferred aspect, the present inventionprovides a nucleic acid which codes for a CDR or set of CDRs or antibodyantigen-binding site or VH domain or VL domain or antibody molecule,e.g. scFv or IgG4, of the invention as defined above.

The present invention also provides constructs in the form of plasmids,vectors, transcription or expression cassettes which comprise at leastone polynucleotide as above.

The present invention also provides a recombinant host cell whichcomprises one or more constructs as above. A nucleic acid encoding anyCDR or set of CDRs or VH domain or VL domain or antigen-binding site orantibody molecule, e.g. scFv or IgG4 as provided, itself forms an aspectof the present invention, as does a method of production of the encodedproduct, which method comprises expression from encoding nucleic acidtherefor. Expression may conveniently be achieved by culturing underappropriate conditions recombinant host cells containing the nucleicacid. Following production by expression a VH or VL domain, or specificbinding member may be isolated and/or purified using any suitabletechnique, then used as appropriate.

Specific binding members, VH and/or VL domains, and encoding nucleicacid molecules and vectors according to the present invention may beprovided isolated and/or purified, e.g. from their natural environment,in substantially pure or homogeneous form, or, in the case of nucleicacid, free or substantially free of nucleic acid or genes origin otherthan the sequence encoding a polypeptide with the required function.Nucleic acid according to the present invention may comprise DNA or RNAand may be wholly or partially synthetic. Reference to a nucleotidesequence as set out herein encompasses a DNA molecule with the specifiedsequence, and encompasses an RNA molecule with the specified sequence inwhich U is substituted for T, unless context requires otherwise.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, mammalian cells, plant cells, insect cells, fungi, yeast andtransgenic plants and animals. Mammalian cell lines available in the artfor expression of a heterologous polypeptide include Chinese hamsterovary (CHO) cells, HeLa cells, baby hamster kidney cells, NSO mousemelanoma cells, YB2/0 rat myeloma cells, human embryonic kidney cells,human embryonic retina cells and many others. A common, preferredbacterial host is E. coli.

The expression of antibodies and antibody fragments in prokaryotic cellssuch as E. coli is well established in the art. For a review, see forexample Plückthun, A. Bio/Technology 9: 545-551 (1991). Expression ineukaryotic cells in culture is also available to those skilled in theart as an option for production of a specific binding member for exampleChadd H E and Chamow S M (2001) 110 Current Opinion in Biotechnology 12:188-194, Andersen D C and Krummen L (2002) Current Opinion inBiotechnology 13: 117, Larrick J W and Thomas D W (2001) Current Opinionin Biotechnology 12:411-418.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Vectors may be plasmids, viral e.g.'phage, or phagemid, or adenoviral, AAV, lentiviral, etc. asappropriate. For further details see, for example, Molecular Cloning: ALaboratory Manual, 3rd edition, Sambrook and Russell, 2001, Cold SpringHarbor Laboratory Press. Many known techniques and protocols formanipulation of nucleic acid, for example in preparation of nucleic acidconstructs, mutagenesis, sequencing, introduction of DNA into cells andgene expression, and analysis of proteins, are described in detail inCurrent Protocols in Molecular Biology, Second Edition, Ausubel et al.eds., John Wiley & Sons, 1986, Short Protocols in Molecular Biology: ACompendium of Methods from Current Protocols in Molecular Biology,Ausubel et al. eds., John Wiley & Sons, 4th edition 1999. Thedisclosures of Sambrook et al. and Ausubel et al. (both) areincorporated herein by reference.

Thus, a further aspect of the present invention provides a host cellcontaining nucleic acid as disclosed herein. Such a host cell may be invitro and may be in culture. Such a host cell may be in vivo. In vivopresence of the host cell may allow intracellular expression of thespecific binding members of the present invention as “intrabodies” orintracellular antibodies. Intrabodies may be used for gene therapy(Marasco W A (1997) Gene Therapy, 4(1): 11).

A still further aspect provides a method comprising introducing suchnucleic acid into a host cell. The introduction may employ any availabletechnique. For eukaryotic cells, suitable techniques may include calciumphosphate transfection, DEAE-Dextran, electroporation, liposome-mediatedtransfection and transduction using retrovirus or other virus, e.g.vaccinia or, for insect cells, baculovirus. Introducing nucleic acid inthe host cell, in particular a eukaryotic cell may use a viral or aplasmid based system. The plasmid system may be maintained episomally ormay incorporated into the host cell or into an artificial chromosome(Csonka E et al. (2000) Journal of Cell Science, 113: 3207-3216;Vanderbyl S et al. (2002) Molecular Therapy, 5(5: 10. Incorporation maybe either by random or targeted integration of one or mere copies atsingle or multiple loci. For bacterial cells, suitable techniques mayinclude calcium chloride transformation, electroporation andtransfection using bacteriophage.

The introduction may be followed by causing or allowing expression fromthe nucleic acid, e.g. by culturing host cells under conditions forexpression of the gene.

In one embodiment, the nucleic acid of the invention is integrated intothe genome (e.g. chromosome) of the host cell. Integration may bepromoted by inclusion of sequences which promote recombination with thegenome, in accordance with standard techniques.

The present invention also provides a method which comprises using aconstruct as stated above in an expression system in order to express aspecific binding member or polypeptide as above.

The nucleic acid molecules of the instant invention can be administeredto a patient in need thereof via gene therapy. The therapy may be eitherin vivo or ex vivo. In a preferred embodiment, nucleic acid moleculesencoding both a heavy chain and a light chain are administered to apatient. In a more preferred embodiment, the nucleic acid molecules areadministered such that they are stably integrated into chromosomes of Bcells because these cells are specialized for producing antibodies. In apreferred embodiment, precursor B cells are transfected or infected exvivo and re-transplanted into a patient in need thereof. In anotherembodiment, precursor B cells or other cells are infected in vivo usinga virus known to infect the cell type of interest. Typical vectors usedfor gene therapy include liposomes, plasmids and viral vectors.Exemplary viral vectors are retroviruses, adenoviruses andadeno-associated viruses. After infection either in vivo or ex vivo,levels of antibody expression can be monitored by taking a sample fromthe treated patient and using any immunoassay known in the art ordiscussed herein. Methods of utilizing an anti-TGFβ antibody in genetherapy are known in the art. See, for example, U.S. Pat. No. 5,824,655(Border), which is incorporated herein by reference in its entirety.

In a preferred embodiment, the gene therapy method comprises the stepsof administering an isolated nucleic acid molecule encoding the heavychain or an antigen-binding portion thereof of an anti-TGFβ antibody andexpressing the nucleic acid molecule. In another embodiment, the genetherapy method comprises the steps of administering an isolated nucleicacid molecule encoding the light chain or an antigen-binding portionthereof of an anti-TGFβ antibody and expressing the nucleic acidmolecule. In a more preferred method, the gene therapy method comprisesthe steps of administering of an isolated nucleic acid molecule encodingthe heavy chain or an antigen-binding portion thereof and an isolatednucleic acid molecule encoding the light chain or the antigen-bindingportion thereof of an anti-TGFβ antibody of the invention and expressingthe nucleic acid molecules.

Dose-correction across species generally requires an adjustment forbody-weight only, if the active agent is an antibody acting in or closeto the vascular system. Effective doses of the antibodies of theinvention have been 0.5-5 mg/kg in rat and mouse in the acute setting.Therefore, for long-term dosing, 0.3-10 mg/kg administered on thehalf-life (expected to be in the region of 21 days in humans) isconsidered likely. Preferable doses are sufficient for efficacy, but lowenough to facilitate optimal administration. For example a dose of lessthan 50 mg facilitates subcutaneous administration. Intravenousadministration is preferable in early clinical trials and may be used asthe route of delivery for severe diseases if the dose is high and thedosing interval long. Subcutaneous injection is generally moreconvenient than intravenous delivery, because it allowsself-administration. However, subcutaneous injection has the potentialto augment any immune response to product. Local administration forlocalized disease can minimize the amount of product required andmaximize the concentration at the site of action. A significant safety(therapeutic window) advantage may be conferred by local administration,avoiding any potential side effects that may develop from chronicsystemic administration.

Further aspects and embodiments of the present invention will beapparent to those skilled in the art in the light of the presentdisclosure, including the following experimental exemplification. Alldocuments referred to anywhere in this specification are incorporated byreference.

Example 1 Generation of Anti-TGFβ ScFvs ScFv Naive Antibody Libraries

A large single chain Fv (scFv) human antibody library derived fromspleen lymphocytes from 20 donors and cloned into a phagemid vector(Hutchings et al., 2001) was used for selections.

ScFv Guided Selection Libraries

A 1D11.16 VH-human VL library was constructed and used to selectmouse-human chimeric antibodies with the desired binding properties. Thehuman light chains from these chimeric antibodies were then cloned intohuman VH-VL and human VH (1D11 CDR3)-VL acceptor libraries. Theselibraries were screened for human antibodies with the desired bindingproperties.

Selection of ScFv Phage Libraries

Recombinant human TGFβ1 and TGFβ2 were supplied by Genzyme Corp.(Framingham, Mass.) and TGFβ3 was purchased from R&D Systems.

ScFvs which recognised TGFβ were isolated from scFv guided selectionlibraries following a series of repeated selection cycles on recombinanthuman TGFβ essentially as described in Vaughan et al. (1996). In brief,following incubation with the library, the immobilised antigen, whichhad been pre-coupled to paramagnetic beads, and bound phage wererecovered by magnetic separation whilst unbound phage were washed away.Bound phage was then rescued as described by Vaughan et al. (1996) andthe selection process repeated.

Selections were performed using TGFβ1, TGFβ2 or TGFβ3 coupled toDynabeads M-270 amine (Dynal) according to the manufacturer'srecommendations. Alternatively, selections used biotinylated TGFβ1 orTGFβ2 prepared using the primary amine specific reagentsuccinimidyl-6-(biotinannido) hexanoate following the manufacturer'sinstructions (EZ link NHS LC Biotin, Pierce).

Outputs from selections were tested as periplasmic preparations in highthroughput screens based on competition assays which measured theability of the scFvs present in the periplasmic preparation to competewith 1D11.16 or the recombinant human TGFβ soluble receptor II-Fcchimaera (sRII, R&D Systems) for binding to TGFβ.

Samples that competed with 1D11.16 or sRII in the high throughputscreens were subjected to DNA sequencing as described in Vaughan et al.(1996) and Osbourn et al. (1996). Clones were expressed and purified asscFvs or IgGs and assessed for their ability to neutralise TGFβs in theMLEC and/or the NHLF assays as described in Examples 4 and 5respectively. Purified scFv preparations were prepared as described inExample 3 of WO 01/66754. Protein concentrations of purified scFvpreparations were determined using the BCA method (Pierce). Purified IgGpreparations were prepared as described below in Example 3.

Example 2 Optimisation of Anti-TGFβ scFvs

ScFvs binding and neutralising TGFβ were generated as described inExample 1. The neutralisation potencies of these antibodies wereincreased on TGFβ3 and/or TGFβ2 and/or TGFβ3 using DNA mutagenesisand/or combinatorial techniques. Antibodies with significantly improvedpotencies on TGFβ1 and/or TGFβ2 and/or TGFβ3 were generated by selectingand screening phage antibody libraries essentially as described inExample 1. The scFvs generated were compared to 1D11.16 in the MLECproliferation assay.

Particular germlines were found to be highly represented amongst thepopulation of high potency, TGFβ-neutralising scFvs. These wereDP-10/1-69 and DP-88/1-e (both members of the VH1 germline family) forthe heavy chain, and DPK22/A27 (V_(K)3 family) for the light chain.These germlines appear to provide a structural framework particularlysuitable for high potency, TGFβ pan-neutralising antibodies. This wasnot predictable, since 1D11.16 VH gene segment is closest to the humangermline DP-7 and the 1D11.16 VL gene segment is closest to the humangermline L16.

PET1073G12, PET1074B9 and PET1287A10 scFvs showed potencies approachingor exceeding those of 1D11.16 on all three TGFβ isoforms in the MLECproliferation assay.

The derived amino acid sequences of PET1073G12, PET1074B9 and PET1287A10VH and VL gene segments were aligned to the known human germlinesequences in the VBASE database (Tomlinson et al., 1997) and the closesthuman germline identified by sequence similarity. The closest humangermline gene for the VH gene segment of PET1073G12 and PET1074B9 wasidentified as DP-10/1-69 (VH1 germline family) and the closest humangermline gene for the VH gene segment of PET1287A10 was identified asDP-88/1-e (VH1 germline family). The closest human germline gene for theVL gene segment of PET1073G12, PET1074B9 and PET1287A10 was identifiedas DPK22/A27 (V_(K)3 germline family). Site directed mutagenesis wasused to change framework residues that differed from germline to thegermline residue, provided that such changes did not produce a loss ofpotency in the MLEC proliferation assay of more than three-fold in theresulting antibody on any TGFB. isoform. If such a loss of potency wasobserved, the non-germline framework amino acid was kept in the finalantibody.

In germlined PET1073G12 and germlined PET1074B9 all framework residuesare germline except for two residues in VH and one residue in VL. Theamino acid sequences for germlined PET1073G12 are described in SEQ IDNO: 2 for VH and SEQ ID NO: 7 for VL. The amino acid sequences forgermlined PET1074B9 are described in SEQ ID NO: 12 for VH and SEQ ID NO:17 for VL.

In germlined PET1287A10 all VH and VL framework residues are germline.The amino acid sequences for germlined PET1287A10 are described in SEQID NO: 22 for VH and SEQ ID NO: 27 for VL.

Example 3 Production of IgG4s

The germlined scFvs PET1073G12, PET1074B9 and PET1287A10 were convertedfrom the scFv format to the IgG4 format by sub-cloning their VH and VLdomains into vectors expressing whole antibody heavy and light chainsrespectively. The VH gene segment was amplified from the pCantab6 scFvexpressing vector and cloned into the pEU8.1(+) vector containing thehuman γ⁴ heavy chain constant domains and regulatory elements to expressthe whole heavy chain in mammalian cells. Similarly, the VL gene segmentwas amplified from the pCantab6 scFv-expressing vector and cloned intothe pEU3.1(−) vector containing the human κ light chain constant domainsand regulatory elements to express the whole light chain in mammaliancells. The pEU3.1(−) and pEU8.1 (+) vectors were based on the vectorsdescribed by Persic et al.(1987) and were modified to introduce the oriPsequence to increase the yields of antibody produced (Shen. et al.,1995; Langle-Rouault et al., 1998). Following cloning, the VH and VLdomains of all three antibodies were sequenced to confirm that nomutations had been introduced during the cloning procedure.

Vectors for the expression of PET1073G12, PET1074B9 and PET1287A10 heavyand light chain were transfected into EBNA-293 cells (Invitrogen).Following gene expression and secretion in the cell supernatant,PET1073G12, PET1074B9 and PET1287A10 IgG4s were purified by protein Aaffinity chromatography (Amersham). The purified antibody preparationswere sterile filtered and stored at 4° C. in phosphate buffered saline(PBS) prior to evaluation. The concentration of IgG was determinedspectrophotometrically using an extinction coefficient based on theamino acid sequence of the IgG as described in Mach et al. (1992). Thepurified IgG were analysed by SEC-HPLC using a Biosep-SEC-S2000 column(Phenomenex) to check for aggregation or degradation of the protein.Reformatted human IgG4 whole antibodies were compared to the 1D11.16antibody in the MLEC and NHLF cell based assays as described in Examples4 and 5 respectively.

Example 4 Neutralisation Potency of Anti-TGFβ Antibodies in the TGFβDependent MLEC Proliferation Assay

The neutralisation potency of purified antibody preparations againsthuman TGFβ bioactivity was assessed using the Mink Lung Epithelial Cell(MLEC) proliferation assay.

The MLEC proliferation assay is based on an assay described byDanielpour et al. (1989a). This assay works on the principle that whenTGFβ1, TGFβ2 or TGFβ3 is added to mink lung epithelial cells this causesan inhibition of the serum induced cell proliferation. Antibodies weretested for neutralisation of TGFβ1, TGFβ2 or TGFβ3 resulting in therestoration of the cell proliferation. Proliferation was measured by theuptake of [³H]-thymidine. The potency of the antibody was defined as theconcentration of the antibody that neutralised a single concentration ofTGFβ1, TGFβ2 or TGFβ3 at a level of 50% (IC₅₀) in nM.

MLEC Proliferation Assay Protocol Plating of MLEC

The MLEC line was obtained from the American Type Culture Collection(Cat.#CCL-64). Cells were grown in Minimum Essential Media (MEM, Gibco)containing 10% FBS (Gibco), 1% penicillin/streptomycin (Gibco) and 1%MEM non essential amino acids solution (Gibco). Confluent cells fromT-175 flasks were dissociated from the flask, spun down, washed, andresuspended in MLEC assay media that was made of MEM containing 1% FBS,1% penicillin/streptomycin and 1% MEM non essential amino acidssolution. An aliquot of the cells was then labelled with trypan blue,counted on a haemocytometer and the cell stock diluted to 1.75×10⁵ cellper ml using assay media. 100 μl of this suspension was added to eachwell of a tissue culture flat-bottomed 96 well plate and incubated for 3to 5 hours.

Preparation of TGFβ/Antibody Solutions

Working solutions of TGFβ1, TGFβ2 or TGFβ3 at 6 ng/ml (6 times the finalassay concentration) and antibodies (including controls such as 1D11.16)at 3 times the final maximum assay concentration were prepared in MLECassay media. The final concentration of TGFβ in the assay (1 ng/ml or 40pM) corresponded to the concentration that induced approximately 80%inhibition of cell proliferation compared to the control with no TGFβ(i.e. EC₈₀ value).

Dilution Plate Set Up

Samples of test and control antibodies were titrated in 3-fold dilutionsteps in MLEC assay media and incubated in the presence and absence ofTGFβ1, TGFβ2 or TGFβ3. All relevant controls were included in everyexperiment: testing of the 1D11.16 and/or reference antibody asappropriate and performing TGFβ1, TGFβ2 or TGFβ3 titrations. Completedplates were left in a humidified tissue culture incubator for 1 hour±15minutes.

Addition of TGFβ/Antibody Solutions to the Plated Cells

After the appropriate incubation times, 100 μl from each well of thedilution plates were transferred to the plated MLEC and the platesreturned to the incubator for 44±2 hours.

Addition of [³H]-Thymidine

25 μl of 10 μCi/m1 [³H)-thymidine (diluted in PBS) was added to each ofthe wells (0.25 μCi/well). The plates were then returned to theincubator for 4 hours±30 minutes.

Cell Harvesting

100 μL of trypsin-EDTA (0.25%, Gibco) was added to each well, platesincubated for 10 minutes in the incubator and cells were harvested usinga Tomtec or Packard 96 well cell harvester.

Data Accumulation and Analysis

Data from the harvested cells were read using a beta-plate reader(TopCount, Packard). Data were analysed to obtain IC₅₀ and standarddeviation values. IC₅₀ values were obtained by using the Prism 2.0(GraphPad) software.

Results

Purified PET1073G12, PET1074B9 and PET1287A10 germlined IgG4s weretested alongside 1D11.16 in the MLEC proliferation assay. IgG4s wereproduced as described in Example 3. Arithmetic mean IC₅₀s±standarddeviation (where IC₅₀ is the concentration of antibody required toneutralise 40 pM TGFβ1, TGFβ2 or TGFβ3 by 50%) are shown in Table 1.

Mean IC₅₀ data for PET1073G12 and PET1287A10 IgG4s shows that theseantibodies have potencies similar or approaching those of 1D11.16 onTGFβ1, TGFβ2 and TGFβ3.

Mean IC₅₀ data suggests that PET1074B9 IgG4 is significantly more potenton TGFβ1 (although a full dose response curve was not obtained in theMLEC assay). As a means for comparison, 1D11.16 showed 12%neutralisation on TGFβ1 at a concentration of 91 pM and PET1074B9 showed78% neutralisation at a similar concentration of 92 pM. Furthermore,PET1074B9 was also tested alongside 1D11.16 in a normal human lungfibroblast assay (NHLF) fibronectin production assay (Example 5). Theresults obtained in the NHLF assay confirm those obtained in the MLECassay: PET1074B9 has potencies similar to those of 1D11.16 on TGFβ2 andTGFβ3 and PET1074(9 is more potent than 1D11.16 on TGFβ1.

Example 5 Neutralisation Potency of Anti-TGFβ Antibodies in the TGFβ3Dependent NHLF Cell Assay

The neutralisation potency of purified antibody preparations againsthuman TGFβ bioactivity was assessed using the Normal Human LungFibroblast (NHLF) fibronectin production assay. This assay measures theability of antibodies to neutralise the production of the extracellularmatrix (ECM) glycoprotein, fibronectin. TGFβs are potent stimulators offibronectin production in cultured fibroblasts (Ignotz and Massaoue,1986) exerting their effects via activation of the c-Jun N-terminalkinase pathway (Hocevar et al., 1999).

NHLF Cell Assay Protocol

NHLF cells were obtained from Clonetics™ and maintained in completefibroblast growth media-2 (FGM-2) in a humidified atmosphere containing5% CO₂ at 37° C. At 90-100% confluence, fibroblasts were plated(1.5×10⁵/well, 24 well format) in 1.5 ml FGM-2 media and allowed toattach for 24 hours at 37° C. Cells were washed with serum-freefibroblast basal media (FBM) and serum starved overnight in 1.5 ml FBMsupplemented with human insulin (100 μg/ml), gentamicin/fungizone (50μg/ml) and ascorbic acid (50 (μg/ml) and incubated for 24 hours at 37°C. All experiments were performed on cells between passage three to six.

Preparation of TGFβ and Antibody Solutions

Working solutions of TGFβ1, TGFβ2 or TGFβ3 at 25 ng/ml (1 nM) andantibodies (including controls such as 1D11.16) were prepared in assaymedia. The final concentration of TGFβ in the assay (250 pg/ml or 10 pM)corresponded to the concentration that induced approximately 80%stimulation of fibronectin production compared to control with no TGFβ(i.e. EC₈₀ value).

Dilution Plate Set Up

Samples of test and control antibodies were serially diluted in 10-folddilution steps in assay media and preincubated in the presence andabsence of TGFβ1, TGFβ2 or TGFβ3 32 for 30 minutes. NHLF cells wereincubated in 2 ml/well assay media for 48 hours at 37° C. After 48 hoursa 0.5 ml aliquot of culture media supernatant was taken for fibronectinanalysis by ELISA.

Human fibronectin ELISA

Fresh or frozen (−20° C.) NHLF supernatant samples were analysed using aTechnoclone™ human fibronectin antigen ELISA kit comprising of a humananti-fibronectin capture monoclonal antibody (clone 6FN) and a HRPconjugated monoclonal anti-fibronectin secondary antibody. Themethodology was as follows:

Nunc-Immuno™ Maxisorp™ 96 well plates were coated (1 μg/well) with ananti-fibronectin capture antibody in coating buffer (12 mM Na²CO3, 35 mMNaHCO³, 0.01% (w/v) thimerosal in distilled water, pH 9.6) for 16 hoursat 4° C. The capture antibody was removed and each well blocked with 100μl of dilution buffer (1% (w/v) BSA in PBS) for 1 hour at 37° C. Afterwashing three times with wash buffer (250 μg/well of 0.5% (v/v) Tween 20in PBS), human plasma fibronectin standards and samples were added tothe plate and incubated for 1 hour at 37° C. The plate was then washedthree times and incubated with an anti-fibronectin HRP secondaryantibody (100 μg/well in dilution buffer) for 30 minutes at 37° C. Theplate was washed three times with wash buffer and 100 μg/well oftetramethylbenzidine (TMB) substrate was added to the plate. Afterincubation of the plate at room temperature for 20 minutes, the reactionwas stopped with 100 μg/well 2 M sulphuric acid. The absorbance at 450nm was then measured using a Dynex MRX plate reader.

Data Analysis

Data are presented as a percentage of the control response to the TGFβisoform under test (100%). Geometric mean pIC50 values and 95%confidence limits were estimated using four-parameter logistic curvefitting (Prism 2, GraphPad Software, San Diego, USA). When a fourparameter fit failed, three or two parameter fit was performed byholding the curve top and/or bottom values constant.

Results

Purified PET1073G12, PET1074B9 and PET1287A10 germlined IgG4s weretested alongside 1D11.16 in the NHLF fibronectin production assay. IgG4swere produced as described in Example 3. Arithmetic mean IC₅₀s±standarddeviation (where IC₅₀ is the concentration of antibody required toneutralise 10 pM TGFβ1, TGFβ2 or TGFβ3 by 50%) are shown in Table 2.

Example 6 Potency in IL-11 Induction Assay

We assessed the neutralisation potency of purified antibody preparationsagainst human TGFβ bioactivity using a A549 cell (human lung epithelialcarcinoma cells) IL-11 induction assay.

We maintained A549 cells (ATCC, Part #: CCL-185) in Growth/Assay Media(435 mL DMEM, 50 mL fetal bovine serum (FBS), 5 mLpenicillin/streptomycin, 5 mL Modified Eagle Medium non-essential aminoacids, 5 mL 100× L-glutamine; all Gibco/Invitrogen). At approximately90% confluence, we plated the cells in 100 μL Growth/Assay Media andallowed cells to attach for 24 hours at 37° C., 5% CO₂.

Preparation of TGFβ and Antibody Solutions

We prepared working solutions of TGFβI (1.8 ng/ml), TGFβ2 (4.2 ng/ml) orTGFβ3 (4.2 ng/ml) and antibodies (including controls) in Growth/Assaymedia.

Dilution Plate Set Up

We serially diluted samples of test and control antibodies in 5-fold(TGFβ2 or TGFβ3) or 10-fold (TGFβ1) dilution steps in Growth/Assay mediaand preincubated in the presence and absence of TGFβ1, TGFβ2 or TGFβ3 at37° C. for 75 minutes. We incubated A549 cells in 200 μl/well assaymedia for 18-24 hours at 37° C. After 18-24 hours, a 100 μl aliquot ofculture media supernatant was taken for IL-11 analysis by ELISA.

Example 7

To determine the biologic efficacy of a human pan-neutralizing TGF-βmonoclonal antibody for treating chronic renal disease and otherclinical indications characterized by pathogenic fibrosis, we studiedthe effect of the antibody in a rat unilateral ureteral obtruction (UUO)model.

Adult Sprague Dawley rats (Taconic Farms, Germantown, N.Y.) weighing250-280 gram (about 6 weeks) were housed in an air-, temperature-, andlight-controlled environment. Rats undergoing UUO received a smallventral midline abdominal incision to expose the left kidney and upperureter. We ligated the ureter at the level of the lower pole of thekidney with silk suture and a second time at about 0.2 cm below thefirst one. Sham operated rats received the same surgical protocol butwithout ureteral ligation.

The obstructed rats were treated with PBS, a murine pan-neutralizingmonoclonal antibody (1D11), an isotype-matched control antibody (13C4)or a human pan-neutralizing TGF-β monoclonal antibody of the inventionas follows. We administered the antibodies to the rats intraperitoneallybeginning on the day of ureteral ligation for a course of 3 weeks. 13C4and 1D11 were administered at 5 mg/kg (3 times/week) and the humanpan-neutralizing antibody was given to the rats at 5 mg/kg (every 5days). At the end of 3 weeks, we sacrificed the rats, perfused thekidneys with PBS for 3 minutes and harvested the perfused kidneys forthe analysis of mRNA, determination of collagen content and histologicalexamination.

To assess the extent of tissue fibrosis, we determined total tissuecollagen content by biochemical analysis of hydroxyproline inhydrolysate extracts according to Kivirikko et al. This assay is basedon the observation that essentially all hydroxyproline in animal tissuesis found in collagen.

We also performed a Sircol collagen assay for total collagen content.The Sircol collagen assay measures the amount of total acid/pepsinsoluble collagens based on the specific binding of Sirius red dye withthe side chain of tissue collagen.

The UUO rats treated with the human pan-neutralizing monoclonal antibodyshowed a 43.4% reduction in hydroxyproline content (1.98±0.26 μg/mg drytissue) when compared to the PBS treated group (3.5±0.3 μg/mg drytissue, p<0.05). The lessening in renal fibrosis was further supportedby the reduction in total solubilized collagen in the affected kidneys,as determined by a Sirius red dye based assay (sham: 18.5±2.6, PBS:69.3±3.8, and human pan-neutralizing monoclonal antibody: 35.6±5.2μg/100 mg tissue, p<0.05 vs. PBS).

We also assessed the ability of a human pan-neutralizing anti-TGF-βmonoclonal antibody to reduce tissue fibrosis by immunohistochemicalexamination.

In control animals, ureteral obstruction for three weeks causedwidespread disruption of renal tubular architecture with markeddistension, cellular atrophy and necrosis/apoptosis, tissue inflammationand tubulointerstitial expansion with evident fibrosis. There was littleevidence of glomerular damage. Rats treated with 1D11 or the humanpan-neutralizing monoclonal antibody, on the other hand, showedpreservation of renal architecture as judged by attenuated tubulardilation and disorganization, reduced inflammatory infiltrates(cellularity) and diminished tubulointerstitial expansion and fibrosis.

We also measured the effect of treatment with a human pan-neutralizinganti-TGF-β monoclonal antibody on TGF-β regulated gene expression.

TGF-β1 mRNA was significantly reduced in the human pan-neutralizingmonoclonal antibody treated UUO animals compared to either PBS-treatedor 13C4 control antibody-treated animals. A significant decrease in mRNAlevels for type III collagen also was seen in the obstructed kidneystreated with the human and murine anti-TGF-β antibodies as compared tothose treated with PBS or 13C4 indicating a decrease in collagensynthesis.

We further confirmed the efficacy of a human pan-neutralizing anti-TGF-βmonoclonal antibody to reduce auto-induced TGF-β synthesis by measuringthe total renal TGF-β1 protein.

Compared to the sham-operated animals, obstructed kidneys exhibited amarked increase in total tissue TGF-β1. Obstructed rats dosed with ahuman pan-neutralizing monoclonal antibody, however, showed 75%reduction of tissue TGF-β1 levels, significantly below the levelsrecorded for both control groups. By comparison, the murine 1D11antibody reduced tissue TGF-β1 levels by 45%, compared to controlgroups.

The above-described results demonstrate that the TGF-β neutralizationwith a human pan-neutralizing anti-TGF-β monoclonal antibody effectivelyinterrupted the TGF-3 autocrine-regulation loop concomitant withprevention of TGF-β1 production and collagen III mRNA expression.

We further determined the effect of a human pan-neutralizing anti-TGF-βmonoclonal antibody on the expression of smooth muscle actin (α-SMA) asan indirect indicator of TGF-β inhibition. Smooth muscle actinexpression is an indicator of activated myofibroblasts, which areassociated with tissue fibrosis and produce fibrous connective tissue.TGF-β is an important inducer of the activation and phenotypictransformation of stromal fibroblasts and resident epithelial cells tomyofibroblastic cells.

We detected α-SMA protein by standard Western blot analysis.

When compared with sham-operated animals, rats with obstructed kidneysshowed dramatic upregulation in α-SMA protein as measured by westernblotting of tissue homogenates (data not shown). Obstructed rats dosedwith a human pan-neutralizing anti-TGF-β monoclonal antibody showedsignificant reduction (75% compared to PBS controls) in measureableα-SMA expression.

These results demonstrate the efficacy of a human pan-neutralizinganti-TGFβ monoclonal antibody in reducing collagen deposition in thefibrotic kidneys, clearly indicating that the antibody is a potentinhibitor of renal collagen production and deposition in this model ofsevere renal injury and tubulointerstitial fibrosis. Because the processof tissue fibrosis in organs such as in lung, liver or kidney possessescommon mechanisms or pathways, the skilled worker will appreciate thatthe antibody is useful in the treatment of chronic renal diseases aswell as other clinical indications characterized by pathogenic fibrosis.

A description of certain preferred claims of the invention follows:

TABLE 1 Geometric Mean IC₅₀ (95% confidence intervals; nM) TGFβPET1073G12 PET1074B9 PET1287A10 1D11.16 isoform n = 6 n = 6 n = 5 n = 25TGFβ1  0.8 <1#   1.8 3.9 (0.3-2.2)  (1-3.6)    (2.9-5.2)  TGFβ2 13.0  1.5 25.0 9.2 (8.7-18.7) (1.1-2.2) (13.3-47.0) (7.3-11.5) TGFβ3  6.0  2.0  0.5 1.0 (4.2-8.4)  (1-4.1)   (0.2-1)    (0.5-2.0) Neutralisation of the anti-proliferative effects of TGFβ1, TGFβ2 andTGFβ3 on MLEC using PET1073G12, PET1074B9 or PET1287A10 germlined IgG4sor 1D11.16. The total number of data points for each mean is indicatedby n number, and represents an independent titration of each antibody.#IC₅₀ values could not be determined as the antibody was too potent inthe concentration range tested and a full dose response curve was notobtained.

TABLE 2 Geometric Mean IC₅₀ (95% confidence intervals) TGFβ PET1073G12PET1074B9 PET1287A10 1D11.16 isoform n = 5 n = 4 n = 4 n = 6 TGFβ1  0.440.18 0.8  0.4  (0.24-0.82) (0.14-0.28) (0.44-1.3)  (0.19-0.96) TGFβ212.0  4.6  3.9  4.0  (5.4-26)   (1.9-11)   (0.9-16.6)  (1.9-8.3)  TGFβ32.8 0.28 0.45 0.16 (1.27-6.5)  (0.02-3.45) (0.25-0.79) (0.04-0.18)Potencies of PET1073G12, PET1074B9 or PET1287A10 germlined IgG4s or1D11.16 in the NHLF assay. The total number of data points for each meanis indicated by n number, and represents an independent titration ofeach antibody.

1-63. (canceled)
 64. A method of treating pulmonary fibrosis or lungfibrosis with an isolated specific binding member that binds to TGFβ1,TGFβ2, and TGFβ3, said binding member comprising an antigen-bindingdomain of an antibody, wherein the heavy chain of said antibodycomprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 3,an HCDR2 comprising the amino acid sequence of SEQ ID NO: 4, and anHCDR3 comprising the amino acid sequence of SEQ ID NO: 5; and whereinthe light chain of said antibody comprises an LCDR1 comprising the aminoacid sequence of SEQ ID NO: 8, an LCDR2 comprising the amino acidsequence of SEQ ID NO: 9, and an LCDR3 comprising the amino acidsequence of SEQ ID NO:
 10. 65. The method of claim 64, wherein theantibody comprises the PET1073G12 VH domain (SEQ ID NO: 2) and thePET1073G12 VL domain (SEQ ID NO: 7).
 66. The method of claim 65, whereinthe antibody further comprises a human IgG4 constant region.
 67. Themethod of claim 65, wherein the antibody further comprises a human IgG1constant region.
 68. The method of claim 64, wherein the isolatedspecific binding member comprises an isolated single chain Fv moleculecomprising the PET1073G12 VH domain (SEQ ID NO: 2) and the PET1073G12 VLdomain (SEQ ID NO: 7).
 69. The method of claim 64, wherein the antibodycomprises the PET1073G12 VH domain (SEQ ID NO: 2).
 70. The method ofclaim 64, wherein the antibody comprises the PET1073G12 VL domain (SEQID NO: 7).
 71. The method of claim 64, wherein the antibody comprises aheavy chain comprising the PET1073G12 VH domain (SEQ ID NO: 2) and ahuman IgG4 constant region, and a light chain comprising the PET1073G12VL domain (SEQ ID NO: 7) and a human κ light chain constant region. 72.The method of claim 64, wherein the isolated specific binding memberneutralizes human TGFβ1, TGFβ2 and TGFβ3.
 73. The method of claim 64,wherein said heavy chain comprises an FR4 amino acid sequence comprisingthe amino acid sequence in SEQ ID NO:
 31. 74. The method of claim 64,wherein the antibody comprises the PET1073G12 VH domain (SEQ ID NO: 2)with up to 5 framework mutations.
 75. The method of claim 64, whereinthe antibody comprises the PET1073G12 VL domain (SEQ ID NO: 7) with upto 5 framework mutations.